Introduction

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Contraction depends on calcium-induced calcium release (CICR) from the sarcoplasmic reticulum (SR) in mammalian heart cells (Fabiato, 1985). The usual trigger for CICR is Ca²⁺ influx through the L-type Ca²⁺ channel [I_Ca(L)]. Calcium entry through reverse mode operation of the Na⁺/Ca²⁺ exchanger (Nuss and Houser, 1992; Sham et al., 1992; Levi et al., 1994) and through T-type Ca²⁺ channels [I_Ca(T)] also can trigger CICR (Sipido et al., 1998). Calcium entry via T-type channels was a much less efficient trigger than entry via L-type channels, inasmuch as at comparable Ca²⁺ influx, there was less Ca²⁺ release from the SR with I_Ca(T) than with I_Ca(L).

The trigger function of reverse mode Na⁺/Ca²⁺ exchange (I_Na/Ca) relative to I_Ca(L) is debated. For example, reverse mode Na⁺/Ca²⁺ exchange is thought to account for phasic intracellular Ca²⁺ transients and contractions observed at test potentials when I_Ca(L) is reduced by Ca²⁺ channel antagonists. In ventricular myocytes from rat (Wasserstrom and Vites, 1996), rabbit (Levi and Issberner, 1996), and guinea pig (Levi et al., 1996), nifedipine (10-32 µM) suppressed 90 to 99% of I_Ca(L) but did not eliminate either the intracellular Ca²⁺ transient or contractions. Other studies detected a trigger function of reverse mode I_Na/Ca, particularly at very positive potentials, have questioned the physiological role of I_Na/Ca-triggered release of SR Ca²⁺_. In cat ventricular myocytes, reverse mode I_Na/Ca triggered phasic contractions in the presence of either 1 µM verapamil or nifedipine or 0.2 mM Cd²⁺, yet a more important role of I_Na/Ca was to load the SR with Ca²⁺ (Nuss and Houser, 1992). Other studies interpreted similar results at +80 mV in rat ventricular myocytes to indicate that reverse mode I_Na/Ca had slower kinetics to induce CICR (Sham et al., 1992). Verapamil (20 µM) or Cd²⁺ (0.3 mM) only partially reduced contractions when I_Ca(L) was suppressed at a test potential of +2 mV (Vornanen et al., 1994). They considered that reverse mode I_Na/Ca contributed to triggering contraction at 35°C but not at 23°C because of the marked temperature dependence of I_Na/Ca. Although reverse mode Na⁺/Ca²⁺ exchange triggered intracellular Ca²⁺ transients during steady-state block of I_Ca(L) by either 10 µM nifedipine (Grantham and Cannell, 1996) or 20 µM nisoldipine (Sipido et al., 1997), its contribution during an action potential was estimated as small and inefficient. Model calculations indicate that Ca²⁺ influx through L-type channels would reduce Ca²⁺ entry through reverse mode Na⁺/Ca²⁺ exchange. This accords with Na⁺/Ca²⁺ exchange having a variable efficiency such that it can provide a larger Ca²⁺ influx when I_Ca(L) is diminished by Ca²⁺ channel antagonists.

In general, those who report an important trigger function of reverse mode Na⁺/Ca²⁺ exchange have used rapid solution switching to deliver L-type Ca²⁺ channel antagonists; those who report a low triggering function have used steady-state conditions for I_Ca(L) block. Dihydropyridine (DHP) Ca²⁺ channel antagonists have been favored in these experiments because they are lipid soluble, very potent, and block the channel preferentially in the inactivated state. Channel block by DHPs is a function of drug concentration and assumed to be largely use-independent. However, L-type Ca²⁺ channel block by rapidly applied nifedipine in frog ventricular myocytes includes a small component of use-dependent block (Méry et al., 1996). That rapidly applied DHP antagonists may not be efficient blockers of L-type Ca²⁺ channels was raised in experiments with 20 µM nisoldipine and 10 to 20 µM nifedipine (Sipido et al., 1995). In rat ventricular myocytes, nifedipine (10 µM) blocked I_Ca(L) by ~70% in 2 min and completely at 4 min (Wasserstrom and Vites, 1996).

Consequently, we reexamined the use-independent and use-dependent components of DHP action on mammalian I_Ca(L). We tested nifedipine action on I_Ca(L) as a function of concentration, exposure time, and stimulus number to ascertain the extent and completeness of blockade. The inorganic ligand Cd²⁺ was used to standardize I_Ca(L) block. In some experiments, we also tested the effects of these compounds on contractions to evaluate the trigger functions of I_Ca(L) and I_Na/Ca. A preliminary account of some of these findings has been presented (Shen et al., 1999).

	Materials and Methods

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Isolation of Ventricular Myocytes. Single ventricular myocytes were enzymatically isolated from the hearts of male and female guinea pigs (250-450 g) anesthetized with sodium pentobarbital (30 mg/kg i.p.) and anticoagulated with heparin (1000 I.U. i.p.). The heart was retrogradely perfused with Tyrode's solution for 5 min at a rate of 8 to 10 ml/min through an aortic cannula in a Langendorff apparatus. The composition of Tyrode's solution was 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 0.33 mM NaH₂PO₄, 10 mM HEPES, and 20 mM glucose; pH was adjusted to 7.4 with NaOH. After disruption of the extracellular matrix with collagenase and protease, the enzymes were washed out by perfusion with 50 ml of Recovery solution. Recovery solution contained 130 mM potassium aspartate, 5 mM K₂ATP, 5 mM HEPES, and 20 mM glucose; pH was adjusted to 7.4 with KOH. The ventricles were removed and the cells were dispersed in Recovery solution and kept at 4°C for at least an hour. An aliquot of cell suspension was placed in a recording chamber (500-µl volume) mounted on the stage of an inverted microscope. After 10 min, it was superfused with Tyrode's solution (2 ml/min); the glucose concentration was 10 mM for experiments. Temperature was 35°C.

Electrophysiology. An EPC 7 patch-clamp amplifier (List Electronics, Darmstadt, Germany) was used to deliver voltage pulses in whole-cell mode. Voltage commands and current data acquisition were controlled by an IBM-compatible computer equipped with pClamp software (version 5.5; Axon Instruments, Burlingame, CA) and a Labmaster TL-1 interface (Axon Instruments). Glass capillary electrodes (1.1 mm i.d.; 1.3 mm o.d.) were filled with a pipette solution whose composition was 120 mM potassium aspartate, 30 mM KCl, 5 mM Na₂ATP, 10 mM MgCl₂, and 5 mM HEPES; pH was adjusted to 7.3 with KOH. The resistance was 2 to 4 M. In initial experiments, the pipette was filled with a Cs⁺-rich solution with EGTA containing 135 mM cesium aspartate, 10 mM NaCl, 5 mM MgATP, 5 mM EGTA, and 10 mM HEPES 10; pH was adjusted to 7.3 with CsOH. Accordingly, 10 mM CsCl was added to the bath solution. The pipette was connected to the amplifier by a Ag-AgCl wire, and the tip was gently pushed against the cell surface. Negative pressure was applied to the pipette interior until a gigaohm seal was formed. After the electrode capacitance was compensated electronically in the cell-attached mode, the cell membrane was ruptured by additional negative pressure.

Drugs and Application. Calcium channel-blocking drugs were applied to myocytes by rapid superfusion from a reservoir via solenoid-controlled delivery. The time for complete solution change, estimated from the membrane current response to doubling the extracellular K⁺ concentration, was <1 s with a t_1/2 of ~200 ms. The applied solutions were warmed and the outlet of the rapid solution device brought within 50 µm of the cell. After recording conditions for the cell had stabilized, the rapid solution device was turned on and Tyrode's solution, identical with the bath solution, applied to the cell. The temperature at the cell's position transiently changed by 0.2-0.3°C when first switching on the Tyrode's solution and by 0.2°C when switching from Tyrode's solution to a test solution. Nifedipine (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide and prepared fresh daily from the stock solution. All nifedipine solutions were protected from light during preparation, storage, and use.

Cell Contraction. A video-edge detector system (Crescent Electronics, Sandy, UT) tracked cell edge motion. A microscope-magnified (400×) cell image was continuously observed on a high-resolution TV monitor via a sequential scanning video camera attached to a side port of the microscope. The camera position was rotated so that the video monitor raster lines were parallel with the long axis of the cell. The video dimension analyzer monitored a selected raster line for light intensity differences between the end of the myocyte and the surrounding field. The signal from the detector was sent to a strip chart recorder and to a videocassette recorder for storage and off-line analysis.

Data Analysis. Steady-state I_Ca(L) block by nifedipine could be readily measured in the presence of Cs⁺-containing solutions. In this case, the extent of block was taken as absolute peak inward current. When K⁺-containing solutions were used, I_Ca(L) block by nifedipine was standardized against that caused by 0.1 to 0.3 mM Cd²⁺. The latter has been shown to block I_Ca(L) completely (Hobai et al., 1997). Measurements are reported as mean ± S.E.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Concentration-Dependent Block of I_Ca(L) by Nifedipine in Steady State

Initially, we determined the concentration-dependent block of I_Ca(L) by nifedipine in the absence of K⁺ currents (see Materials and Methods). Membrane voltage was stepped from 80 to 40 mV for 350 ms to inactivate the fast Na⁺ and the I_Ca(T) currents. A second voltage jump to +10 mV for 300 ms elicited I_Ca(L); the clamp protocol was repeated at 0.1 Hz. Block of I_Ca(L) by Cd²⁺ (0.1, 0.3, 1.0 mM) appeared complete because the peak of early inward current was positive after Cd²⁺ application for at least 2 min. The average Cd²⁺-sensitive currents of eight cells are essentially equal and amounted to 854 ± 126 (0.1 mM), 857 ± 131 (0.3 mM), and 856 ± 133 pA (1 mM). The effect of 0.1 mM Cd²⁺ was taken as the standard for 100% block of I_Ca(L).

Nifedipine (0.1-100 µM) was cumulatively applied to the same myocyte; each concentration was present for at least 3 min. Half-maximal inhibition (IC₅₀) of I_Ca(L) occurred at 0.3 µM nifedipine when the holding potential was 80 mV between test pulses (Fig. 1, filled squares). In steady state, nifedipine blocked I_Ca(L) by 94 ± 2.3 (30 µM) and 99 ± 0.8% (100 µM), respectively. When nifedipine was applied at single rather than cumulative concentrations, I_Ca(L) block averaged 91 ± 3.5% for 30 µM (n = 5 cells) and 99 ± 4.0% with 100 µM nifedipine (n = 4 cells). A mixture of 30 µM nifedipine plus 30 µM Cd²⁺ (Nif 30/Cd 30) blocked I_Ca(L) by 97 ± 1.6% (n = 4 cells). The IC₅₀ for nifedipine block of I_Ca(L) decreased to 50 nM when the holding potential was maintained at 40 mV between test pulses (Fig. 1, filled circles).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent block of ICa(L) by nifedipine in steady state. Pipette and bath solutions contain Cs+. Two-step voltage-clamp pulses (see text for details) were applied at 0.1 Hz. The curve conforms to a 1:1 binding relation between nifedipine and receptor; the IC50 for nifedipine was 0.3 µM when voltage was held at 80 mV between test pulses (). The IC50 for nifedipine was reduced to 0.05 µM when the membrane was held at 40 mV ().

Steady-state inactivation of I_Ca(L) was determined in the absence and presence of 0.3 µM nifedipine (n = 4 cells). A 1-s command changed the conditioning potential from 80 to +10 mV in 10-mV steps. Afterward, a 10-ms return to 40 mV was inserted before applying a 200-ms jump to the test potential of +10 mV. Inactivation of I_Ca(L) was described by a Boltzmann relation (Fig. 2). Voltage-dependent inactivation of I_Ca(L) at 50% was shifted from 36 ± 3.3 (control) to 53 ± 1.6 mV (nifedipine). The slope factor was 14 ± 1.5 and 15 ± 1.5 in control and nifedipine, respectively. Steady-state block by nifedipine of I_Ca(L) in the presence of K⁺-rich pipette solution was essentially the same as in the presence of Cs⁺-rich pipette solution (vide infra).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Steady-state inactivation of ICa(L) in the absence () and presence of 0.3 µM nifedipine (). The experimental protocol is shown as an inset. Ordinate, ICa(L) as fraction of maximum; abscissa, conditioning membrane potential. The curves are drawn according to Boltzmann relations; see text for details.

Concentration- and Time-Dependent Block of I_Ca(L) by Rapidly Applied Nifedipine and/or Cadmium

Nifedipine Alone. In some studies of excitation-contraction (E-C) coupling (see the Introduction), the ability to suppress I_Ca(L) completely on the first test pulse after rapid application of a blocking agent has been emphasized. Thus, I_Ca(L) block should be maximal and complete on the first test pulse and be invariant during a train of test pulses. The protocol to test this hypothesis included two trains of 200-ms voltage-clamp pulses (40 to +10 mV at 0.5Hz) separated by a 10-s rest interval at 40 mV. The test-blocking agent was applied by rapid superfusion and present throughout the 10-s interval and the second train of test pulses. Membrane voltage was held at 40 mV to promote the blocking effect of nifedipine; the pipette solution was K⁺-rich (see Materials and Methods).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Development of ICa(L) block by nifedipine (30 and 100 µM) or 300 µM Cd2+; K+-rich pipette solution. After 10 conditioning pulses (see inset at top of A for protocol), nifedipine or Cd2+ was applied during a 10-s rest interval and 10 subsequent test pulses. A, membrane currents in one cell in control (0), 30 (1), or 100 µM (2) nifedipine or 300 µM Cd2+ (3) on the 1st (top) and 10th test pulse (bottom) after the 10-s interval. B, summary of results with unblocked ICa(L) (a), use-dependent (b), and use-independent (c) block of ICa(L).

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View larger version (41K): [in this window] [in a new window]   Fig. 4.   Concentration and time dependence for ICa(L) block by nifedipine. Experiments done as in Fig. 3 with K+-rich pipette solution; ICa(L) block standardized with 0.3 mM Cd2+. Each column represents data from increasing application intervals of 5, 10, 15, 20, and 30 s for a nifedipine concentration shown at top. In each column, fractional block by use-independent (unfilled) and use-dependent (filled) actions of nifedipine is shown; N, number of cells at each interval. Unblocked fraction of ICa(L) indicated by cross-hatching.

Nifedipine and Inactivation of I_Ca(L)

Nifedipine increased the rate of I_Ca(L) inactivation (Lee and Tsien, 1983). We evaluated the hypothesis that some block of I_Ca(L) occurs during the first test pulse in experiments with 3 µM nifedipine. The voltage-clamp protocol was the same as described in Fig. 3. In control, I_Ca(L) inactivated in a biexponential manner with time constants (in milliseconds) ₁ and ₂ of 5.5 ± 0.4 and 51.3 ± 6.0 on the 1st test pulse and 5.2 ± 0.4 and 59.0 ± 3.3 on the 10th test pulse, respectively (n = 11 cells). In nifedipine, ₁ decreased by 20% to 4.4 ± 0.5 ms (1st test pulse; P = .006) and by 33% to 3.5 ± 0.4 ms (10th test pulse; P = .002). There was a tendency of ₂ to decrease in nifedipine on the 1st (46.8 ± 3.8 ms) and 10th (53.3 ± 8.2 ms) test pulses, respectively. However, these reductions were not statistically significant (P = .3 and .36, respectively). These averages are from all 11 cells in which ₁ and ₂ were reduced in seven cells. It was difficult to discern two phases of inactivation accurately at higher nifedipine concentrations. We measured the half-time for inactivation (t_1/2) in experiments with 30 µM nifedipine (n = 8 cells) and found that the control t_1/2 was reduced from 11.5 ± 1.8 to 8.5 ± 1.2 ms on the 1st test pulse (P = .01) and from 12.0 ± 2.2 to 8.5 ± 1.2 on the 10th test pulse (P = .01).

Test of Combination of Nifedipine and Cadmium

Cadmium inhibited I_Ca(L) with an IC₅₀ of ~2 µM (Hobai et al., 1997). The kinetics of block by Cd²⁺ is rapid (Lansman et al., 1986) and there is little use-dependent inhibition of I_Ca(L) at 10 s (Fig. 3). After 5-s application, Cd²⁺ inhibited 98 + 2.0% of I_Ca(L) elicited on the first test pulse (n = 13). The residuum of 2% is less than that seen with nifedipine and was abolished at 10-s application intervals (<1%). Thus, 0.3 mM Cd²⁺ inhibited I_Ca(L) nearly completely on the first test pulse at 10 s. Because 0.3 mM Cd²⁺ inhibits I_Na/Ca by almost 50%, it cannot be used to distinguish the triggering roles of I_Ca(L) versus I_Na/Ca in E-C coupling (Hobai et al., 1997).

Inhibition of I_Na/Ca at 30 µM Cd²⁺ is predicted to be 3%, whereas suppression of I_Ca(L) is estimated at 88% by these authors. At 30 µM, Cd²⁺ inhibited I_Ca(L) by 83 ± 2.2 (n = 5), 90 ± 2.2 (n = 6), and 95 ± 1.3% (n = 4) on the first test pulse after 5-, 10-, and 15-s rest intervals, respectively. Inhibition by 30 µM Cd²⁺ amounted to 96 ± 1.2% at the 10th test pulse, which was steady state (n = 15). We tested the effects of Nif 30/Cd 30 to increase the use-independent inhibition of I_Ca(L). On the 1st and 10th test pulses after 10-s application of Nif 30/Cd 30 (Fig. 5A, traces labeled "1"), peak inward current was essentially the same as that seen after 0.3 mM Cd²⁺ alone (Fig. 5A, traces labeled "2"). The inward shift of holding current at 40 mV with 0.3 mM Cd²⁺ may result from I_Na/Ca suppression. The end-of-pulse currents were the same in the presence and absence of the test agents. A summary of experiments after 10-s application is given in Fig. 5B. Nif 30/Cd 30 produced a use-independent inhibition of I_Ca(L) of 95 ± 4.5% at 5-s, 97 ± 1.8% at 10-s, and 96 ± 4.1% at 15-s application. Thus, combining low concentrations of Cd²⁺ and nifedipine produced use-independent inhibition of I_Ca(L) at 10 s that was equivalent to that seen with 100 µM nifedipine.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Concentration and time dependence for ICa(L) block by a mixture of 30 µM nifedipine plus 30 µM Cd2+ (Nif 30/Cd 30). Experimental conditions are as in Fig. 3. A, membrane currents in one cell in control (0), Nif 30/Cd 30 (1) or 300 µM Cd2+ (2) on the 1st (top) and 10th test pulse (bottom) after a 10-s application period. B, summary of results (n = 5 cells) for 10-s application with unblocked ICa(L) (a), use-dependent (b), and use-independent (c) block of ICa(L).

Relationship between Block of I_Ca(L) and Contraction by Test Ligands

Experiments at +10 mV. We repeated the experiments with ligands used to block I_Ca(L) to ascertain the coupling between the L-type Ca²⁺ current and contraction at a test potential of +10 mV. The protocol is shown in the upper portion of Fig. 6B. Six 200-ms conditioning pulses from 80 to 30 mV were applied at 1 Hz to maintain a relatively constant SR Ca²⁺ content. During the 10-s pause, membrane voltage was held at 40 mV; a single 200-ms pulse to +10 mV elicited I_Ca(L) and a contraction. Results from an experiment on a single ventricular myocyte are shown in Fig. 6. The control I_Ca(L) and its corresponding contraction are shown in Fig. 6, A and B, respectively. The current and contraction obtained on the first test pulse after 10-s application of 0.3 mM Cd²⁺ are indicated by the filled squares; both variables are completely suppressed. The records just after washout of Cd²⁺ are not shown. Subsequently, 30 µM nifedipine was tested; I_Ca(L) and its accompanying contraction was greatly, but not completely blocked (filled circles). Like 0.3 mM Cd²⁺, Nif 30/Cd 30 completely blocked the test I_Ca(L) and its contraction on the first test pulse after 10 s. Test contraction recovered essentially completely after washout of Nif 30/Cd 30 (Fig. 6B, bottom). Figure 6A (bottom) shows amplified, superimposed current traces taken from the cell. A transient inwardly directed current is evident in the test of 30 µM nifedipine. In contrast, the current traces at +10 mV in either 0.3 mM Cd²⁺ or Nif 30/Cd 30 do not show this inwardly directed transient. The inward shift of current in 0.3 mM Cd²⁺ is consistent with suppression of I_Na/Ca, which is outward at +10 mV. The end-of-pulse currents at +10 mV are the same in 30 µM nifedipine and Nif 30/Cd 30. 

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Coupling between ICa(L) and contraction in one ventricular myocyte in the absence and presence of L-type Ca2+ channel antagonists. After six conditioning pulses (80 to +30 mV at 1 Hz), membrane was held at 40 mV for 10 s during which time antagonist was rapidly superfused. Test pulse to +10 mV followed. A, ICa(L) in control () and in 300 µM Cd2+ (), or 30 µM nifedipine () or Nif 30/Cd 30 (). Lowest trace in A shows superimposed currents symbolized as above with control as . B, experimental protocol shown above. Cell contraction records taken from control (), 300 µM Cd2+ (), 30 µM nifedipine (), and Nif 30/Cd 30 (). The lowest trace is a record after washout of Nif 30/Cd 30 solution. Washout records between () and () and between () and () not shown.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Summary of blocking effects of test ligands on ICa(L) and contraction. The ordinate shows results as percentage of decrease (mean ± S.E.) of ICa(L) (), percentage of decrease of cell shortening (CS, ), and complete block of CS () grouped according to ligand concentration on the abscissa. Number of cells shown in parentheses. See text for details.

Membrane Current and Cell Contraction at +50 mV

Reverse mode I_Na/Ca increases and I_Ca(L) decreases as membrane voltage becomes more positive. We tested the hypothesis that I_Ca(L) may be present at +50 mV. From a holding potential of 40 mV, membrane voltage was jumped in 10-mV steps to +100 mV at 0.33 Hz before and after addition of 100 µM nifedipine (n = 6 cells). The reversal potential of nifedipine-sensitive I_Ca(L) was 68 ± 2.7 mV, which is comparable to that seen with nisoldipine (Sipido et al., 1997).

We next evaluated the effects of 100 µM nifedipine (Fig. 8A) or Nif 30/Cd 30 (Fig. 8B) on membrane current and contraction at +50 and +100 mV with the same conditioning protocol as in Fig. 6. At +50 mV, the average contraction amplitude was 4.0 ± 0.59 µm (n = 13 cells), essentially the same as at +10 mV (3.6 ± 0.21 µm; n = 38; P = .42). Initial membrane current at +50 mV shifted outward by 120 pA in 100 µM nifedipine (Fig. 8A, left, inset). In seven such experiments, nifedipine shifted peak membrane current at +50 mV outward by 212 ± 30.0 pA (P < .01) yet the end-of-pulse current differed by only 14 ± 15.1 pA (P = .38). There was no significant change in membrane currents at either 40 (14 ± 18.1 pA; P = .48) or 80 mV (9 ± 14.3 pA; P = .81). These results can be explained by block of inwardly directed I_Ca(L) at +50 mV with no effect on I_Na/Ca. In the example shown in Fig. 8A, cell shortening decreased from 3.2 (control) to 1.9 µm in nifedipine; contraction latency increased from 45 to 75 ms. Nifedipine completely blocked contraction at +50 mV in only one cell. In the remaining six cells, cell shortening was reduced from 4.4 ± 0.67 to 2.5 ± 0.67 µm (43% decrease; P = .04) and the latency to contraction onset was 67 ± 4.9 ms in 100 µM nifedipine compared with 48 ± 3.0 ms in control (P = .01).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of nifedipine (100 µM) or Nif 30/Cd 30 on membrane current and contraction at positive potentials. Voltage-clamp conditioning protocol same as in Fig. 5 with 10-s rest interval during which time test drugs were applied by rapid superfusion. In all traces, membrane current is uppermost and cell shortening is lowermost. A, nifedipine action (trace 1) at +50 mV (left) and +100 mV (right) test potentials. B, Nif 30/Cd 30 effects (trace 2) at +50 and +100 mV, respectively. See text for details.

The combination of Nif 30/Cd 30 was tested in the remaining six cells. In the example shown (Fig. 8B, left), peak current shifted outward by 225 pA. Control contraction amplitude and latency were 7.0 µm and 60 ms, respectively; these values changed to 2.2 µm and 100 ms in Nif 30/Cd 30. Nif 30/Cd 30 had the same actions on membrane current as 100 µM nifedipine. Thus, Nif 30/Cd 30 (n = 6) did not change current at 40 mV (12 ± 6.8 pA; P = .72) or at 80 mV (33 ± 18.1 pA; P = .30). However, peak current at +50 mV shifted outward by 218 ± 30.4 pA (P < .01) and not at end of pulse (9 ± 26.7 pA; P = .95). Contractions ceased in three of six cells with Nif 30/Cd 30 and decreased from 3.5 ± 1.0 to 1.2 ± 0.70 µm (66% decrease; P = .01) in the remainder. In these three cells, contraction onset was delayed from 53 ± 11.5 to 107 ± 11.5 ms by Nif 30/Cd 30 (P < .01). Thus, at +50 mV, there is an inwardly directed I_Ca(L) whose block by nifedipine or Nif 30/Cd 30 not only decreased the extent of cell shortening but also delayed the onset of contraction.

Addition of 5 mM Ni²⁺ shifted membrane current inward at +50 and 40 mV and outward at 80 mV. In six experiments with Ni²⁺, membrane current shifted inward by 238 ± 42.2 pA at 40 mV (P < .01) and outward by 158 ± 46.5 pA at 80 mV (P < .01). Thus, Ni²⁺suppressed a current whose reversal potential is between 40 and 80 mV, consistent with its being I_Na/Ca. Nickel had no effect on peak current at +50 mV (33 ± 86.1 pA; P = .19) presumably because the outward shift from I_Ca(L) block was offset by an inward shift due to I_Na/Ca suppression. Evidence for the latter is the inward shift in end-of-pulse current by 273 ± 53.5 pA (P < .01). Contractions were completely eliminated by 5 mM Ni²⁺ (n = 6 cells).

At +100 mV, current through L-type Ca²⁺ channels should be outward and carried by K⁺ (Lee and Tsien, 1983). Nifedipine (100 µM) shifted peak membrane current slightly inward by 35 pA at +100 mV (Fig. 8A, right). Neither contraction amplitude (3.2 µm) nor latency (70 ms) changed in nifedipine. On average, nifedipine shifted peak current inward by 180 ± 44 pA (n = 6 cells). With Nif 30/Cd 30 (Fig. 8B, right), current shifted inward by 100 pA. Cell shortening increased slightly from 5.8 to 6.0 µm, whereas latency remained constant at 70 ms. The Nif 30/Cd 30-sensitive current of 152 ± 38 pA (n = 6) was indistinguishable from that of nifedipine. Before drug addition, the average amplitude of contraction at +100 mV (4.9 ± 0.64 µm; n = 12) is larger than at +50 mV (4.0 ± 0.59 µm), and the latency to onset of the contraction at +100 mV (81 ± 4.5 ms) is greater than at 50 mV (50 ± 2.9 ms). Neither 100 µM nifedipine nor Nif 30/Cd 30 significantly changed contraction amplitude at +100 mV. In six cells, contraction amplitude averaged 4.8 ± 0.80 µm in control versus 4.3 ± 0.80 µm with 100 µM nifedipine (P = .12). In another six cells, cell shortening averaged 5.0 ± 1.0 and 5.3 ± 1.0 µm in control and Nif 30/Cd 30, respectively (P = .14). Contraction onset at +100 mV is slightly delayed by 100 µM nifedipine from 75 ± 6.2 to 88 ± 8.7 ms (P = .08) and by Nif 30/Cd 30 from 87 ± 6.7 to 95 ± 8.8 ms (P = .09), respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Rapidly applied nifedipine displayed use-dependent and use-independent components of I_Ca(L) block. The steady-state IC₅₀ was 0.3 µM at a holding potential of 80 mV and 50 nM when the membrane was held at 40 mV (Lee and Tsien, 1983; Yamamoto et al., 1990). Depolarization promotes I_Ca(L) block because DHP affinity for receptor is greatest as L-type channels shift toward the inactivated state (Carmeliet and Mubagwa, 1998). The V_0.5 for steady-state inactivation of I_Ca(L) shifted by 17 mV to more negative potentials in 0.3 µM nifedipine as predicted (Sunami et al., 1995; for review, see Carmeliet and Mubagwa, 1998).

Use-Independent Block of I_Ca(L). Fractional block of I_Ca(L) on the first test pulse increased with nifedipine concentration and application time, a finding favorable for studying contraction triggering mechanisms. Neutral DHPs rapidly partition into the plasma membrane lipid bilayer (Herbette et al., 1989), binding at hydrophobic amino acids ~11 to 14 Å from the external plasma membrane surface (Bangalore et al., 1994) of the sixth transmembrane segments of domains III and IV in the L-type Ca²⁺ channel 1-subunit (Hockerman et al., 1997). Membrane partitioning did not limit kinetics of nifedipine action in frog ventricular myocytes (Méry et al., 1996).

Use-Dependent Block of I_Ca(L) by Nifedipine. Use-dependent block diminished as nifedipine concentration and time increased, and was least with Nif 30/Cd 30. Use-dependent block by nifedipine after 10 test pulses underestimates the magnitude of this component at steady state. DHPs exert use-dependent block of I_Ca(L) in mammalian and amphibian ventricular myocytes even when present during 3- to 15-min rest intervals (Lee and Tsien, 1983; Uehara and Hume, 1985; Sunami et al., 1995) and when rapidly applied at saturating concentrations (Levi and Issberner, 1996; Méry et al., 1996).

Nifedipine Effects on I_Ca(L) and Contraction at +10 mV. In steady state, nifedipine suppressed the slow inward current and contraction force by 50% at 0.3 and 0.5 µM, respectively (Bayer et al., 1977). We find contraction eliminated when either nifedipine or Nif 30/Cd 30 suppressed I_Ca(L) by 95% on the first test pulse after a 10-s rest exposure. The advantage of Nif 30/Cd 30 is the presence of ligands that use hydrophobic and hydrophilic pathways to their receptor sites. Both ligands are largely use-independent, unlike nifedipine combined with the verapamil analog D600 (Howarth and Levi, 1998); D600 is very use-dependent (for review, see Carmeliet and Mubagwa, 1998).

Nifedipine Effects on Membrane Current and Contraction at +50 and +100 mV. Reverse-mode Na⁺/Ca²⁺ exchange can trigger Ca²⁺ release at voltages 50 mV because disabling the SR with caffeine (Sham et al., 1992) or ryanodine (Sipido et al., 1997) blocks Ca²⁺ release transients at +80 mV (but see Adachi-Akahane et al., 1997; Mattiello et al., 1998).

Limitations. L-type Ca²⁺ channel block by nifedipine was indexed with Cd²⁺. The implications of nonselective block by Cd²⁺ have been presented (vide supra). A second limitation centers around the relative gain of triggering (Stern et al., 1999) and the synergy of triggers (Litwin et al., 1998). Gain (Ca²⁺ release from SR/L-channel Ca²⁺ influx) decreases as membrane voltage approaches E_Ca (Wier et al., 1994; Stern et al., 1999), yet the relative efficacy of I_Ca(L) increases as Ca²⁺ influx is blocked by drugs (Cannell et al., 1995). In contrast, Ca²⁺ entry via reverse mode I_Na/Ca and I_Ca(L) could interact synergistically with the former amplifying the trigger effect of the latter (Litwin et al., 1998). Our findings at +10 mV do not accord with the synergy hypothesis because 95% I_Ca(L) block prevented contractions and contraction latency did not shift at <95% I_Ca(L) block. Nifedipine (100 µM) or Nif 30/Cd 30 delayed residual contractions at +50 mV and had no effect on contraction amplitude and latency at +100 mV. We assume reverse mode I_Na/Ca could trigger Ca²⁺ transients and contractions at these positive potentials as reported by other studies (Nuss and Houser, 1992; Sham et al., 1992; Sipido et al., 1997; Litwin et al., 1998). However, some studies have not detected this outcome (Adachi-Akahane et al., 1998; Mattiello et al., 1998). Nifedipine-resistant contractions at these positive potentials (Fig. 8) did not relax until repolarization such as can occur with tonic entry of Ca²⁺ (Nuss and Houser, 1992; Adachi-Akahane et al., 1997). Experiments that disable the SR release mechanism are needed to distinguish between triggered and tonic nifedipine-resistant contractions at positive potentials. Finally, experiments with action potential-initiated contractions should provide evidence about the physiological role of Ca²⁺ influx via the exchanger on E-C coupling.