Introduction

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There is major clinical interest in antiarrhythmic drugs that prolong APD and are effective in controlling life-threatening ventricular arrhythmias (Colatsky and Follmer, 1989; Katritis and Camm, 1993; Yusuf and Teo, 1991). Unfortunately, most of the drugs that prolong the action potential manifest reverse use-dependence: they lengthen the APD and the refractory period more at slow than at rapid heart rates (Hondeghem and Snyders, 1990; Knilans et al., 1991; Gwilt et al., 1991; Jurkiewicz and Sanguinetti, 1993; Funck-Brentano, 1993). As a result, the drugs lose their antiarrhythmic effectiveness during tachycardia and may induce bradycardia-dependent torsade de pointes (Roden and Hoffman, 1985). Amiodarone has been reported to manifest less reverse use-dependence than other antiarrhythmics (Anderson et al., 1989; Kodama et al., 1992; Sosunov et al., 1996), but it has a unique and incompletely understood mechanism of action. In contrast to those drugs that acutely prolong APD by means of blocking K⁺ channels, or increasing inward Na⁺ current (Lee, 1992), amiodarone prolongs APD only when applied chronically (Gallagher et al., 1989). This effect of amiodarone may be related to a thyroid hormone-mediated mechanism rather than to direct interaction with ionic channels (Talajic et al., 1989; Unger et al., 1993). Moreover, amiodarone therapy is complicated by significant cardiac and extracardiac toxicity (Gill et al., 1992). Thus the development of alternative drugs is of interest.

Nibentan [N-(4-nitrobenzoil)-N-N-diethyl-1-5 pentadiamin hydrochlorid] (C₂₂H₂₉N₃O₃HCl) is a recently synthesized antiarrhythmic agent. It has been reported to prolong APD in canine PF and guinea pig papillary muscle (Rosenshtraukh et al., 1995). The i.v. administration of nibentan to dogs significantly increased atrial and ventricular refractoriness and had no effect on ventricular contractility, blood pressure or atrial and intraventricular conduction (Rosenshtraukh et al., 1995). In addition, this compound prevented ventricular fibrillation after acute coronary artery occlusion (Rosenshtraukh et al., 1995). Moreover, clinical studies have demonstrated nibentan's promising antiarrhythmic efficiency, especially for supraventricular arrhythmias (Maykov et al., 1995; 1996; Ruda et al., 1996). The purpose of the present investigation was to detail more thoroughly the electrophysiologic properties of nibentan, paying special attention to the rate-dependence of its effects on the APD in different cardiac tissues.

	Methods

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Mongrel dogs weighing 10 to 20 kg were anesthetized with sodium pentobarbital (30 mg/kg i.v.). Their hearts were removed through a left lateral thoracotomy and immersed in cold Tyrode's solution equilibrated with 95% O₂/5% CO₂ and containing (mM): NaCl 131, NaHCO₃ 18, KCl 4, CaCl₂ 2.7, MgCl₂ 0.5, NaH₂PO₄ 1.8 and dextrose 5.5. Free-running PF or ventricular myocardial or atrial preparations were dissected from the right and left ventricles or atria, placed in a tissue bath and superfused with Tyrode's solution warmed to 37°C (pH was 7.35 ± 0.05). Solution was pumped through the tissue bath at a flow rate of 12 ml/min, changing chamber content three times per minute. The bath was connected to ground via a 3 M KCl/Ag/AgCl junction.

All preparations were impaled with 3 M KCl-filled glass capillary microelectrodes that had tip resistances of 10 to 20 megohms. V_max was obtained by electronic differentiation with an operational amplifier. The electrodes were coupled by an Ag/AgCl junction to an amplifier with high input impedance and input capacity neutralization. Transmembrane action potentials and V_max were displayed on a digital storage oscilloscope (model 4074, Gould, OH) and stored in digitized form for consequent analysis. For stimulation of preparations, standard techniques were used to deliver square-wave pulses 1.0 ms in duration and 1.5 times threshold via bipolar Teflon-coated silver electrodes.

Nibentan was freshly dissolved in distilled water (5 × 10⁴ M) and then diluted in Tyrode's solution to achieve the desired final concentrations.

Experiments with PF. To investigate frequency-dependence of drug effects, we studied normal, "fast-response" action potentials in fibers driven at CL of 2000, 1000, 500, and 300 ms in sequence. After we obtained control records (after 60 min of stabilization in control Tyrode's solution), the preparations were superfused with Tyrode's containing graded concentrations (1 × 10⁸ through 1 × 10⁶ M) of nibentan. Previous experiments with Purkinje fibers (Rosenshtraukh et al., 1995) and preliminary experiments with myocardial preparations showed that steady-state nibentan effects on action potential parameters were achieved in 30 to 40 min. Therefore, the preparations were allowed to equilibrate for 40 min at each nibentan concentration. The transmembrane potential characteristics recorded were MDP, action potential amplitude, V_max and APD to 50% (APD₅₀) and 90% (APD₉₀) repolarization. The rate of the initial rapid repolarization of the action potential (phase 1 slope) was measured at a rapid sweep speed as the slope of the linear portion of this phase (Knilans et al., 1991). The average rate of repolarization during phase 2 (phase 2 slope) was measured as the slope of the line tangent to maximum and minimum potentials recorded during this phase (Zaza et al., 1989). The rate of repolarization during the terminal phase of repolarization (phase 3 slope) was determined by numeric differentiation of the digitized signal during the terminal phase of repolarization; the maximum value of the derivative was taken.

Experiments with myocardial preparations. Epicardial strips (~1.0 × 1.5 × 0.1 cm) were filleted with a surgical blade from the right and left ventricular free walls. Endocardial strips of the same size were obtained from the surfaces of the papillary muscles and free walls (Litovsky and Antzelevitch, 1993). Atrial strips (~1.0 × 1.5 cm) were removed from left and right atria and placed in a tissue bath, endocardial surface up. The right atrial preparations did not include tissue from the sinus nodal area. After control records were obtained (after 5 to 6 h of stabilization in control Tyrode's solution), the preparations were superfused with Tyrode's containing graded concentrations of nibentan (1 × 10⁸ through 5 × 10⁶ M). The drug effects were studied in preparations driven at CL of 2000, 1000, and 500 ms. Preliminary studies in our laboratory, as well as published studies by us (Rosen et al., 1972; Anyukhovsky and Rosen, 1994) and by others (Wyse et al., 1993) attest to the stability of the various preparations used for periods of exceeding the time frame of our nibentan experiments.

Statistical analysis. Microelectrode data were analyzed from impalements maintained throughout the course of each experimental protocol. Automaticity is reported only for experiments in which the control automatic rates showed a variance not greater than 10%. Data are expressed as mean ± S.E.M. The statistical technique used was analysis of variance for two-factor experiments with repeated or nonrepeated measures and was Bonferroni's test when the F value permitted (Winer et al., 1991). Significance was determined at P < .05.

	Results

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Figure 1 illustrates representative PF transmembrane potentials recorded at a cycle length of 1000 ms. Data summarizing the effects of varying concentrations of nibentan in all experiments at all CL are shown in table 1. The respective data for all types of myocardial action potentials are presented in figure 2 and tables 2, 3 and 4. No significant effect on the MDP was seen. The compound had no effect on the action potential amplitude in either type of ventricular muscle and induced a small but statistically significant decrease in PF and atrial muscle action potential amplitude at the highest concentrations studied. Nibentan produced a modest but significant concentration- and use-dependent decrease in V_max in all tissues. Figure 3A illustrates this effect in PF. Membrane responsiveness was also depressed to a similar extent at all levels of membrane potential (fig. 3B). Nibentan had no effect on the slope of phase 1 of PF action potentials (table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   A representative experiment illustrating the actions of nibentan on the transmembrane action potential of a PF driven at a CL of 1000 ms. In each panel, the top trace shows the transmembrane action potential, and the lower trace shows Vmax. All records were obtained after 40 min of exposure to each concentration of nibentan. Vertical calibration is for the action potential and Vmax; horizontal calibration is for the action potential.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Examples of the effects of nibentan on ventricular subendocardial and subepicardial muscle and atrial muscle action potentials at a CL of 1000 ms. Vertical calibrations are for the action potential and Vmax, respectively; horizontal calibration is for the action potential.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A) Use-dependent blocking effects of nibentan (1 × 107 M and 1 × 106 M) on Vmax of PF action potentials. *P < .05 vs. control at the same CL (n = 8). B) Effects of nibentan (5 × 107 M) on membrane responsiveness of PF driven at a basic CL of 500 ms. *P < .05 vs. control at the same level of membrane potential (n = 6).

The most prominent effect of nibentan was a lengthening of the APD in all myocardial tissues. However, a significant tissue specificity in concentration-dependence of this action was observed. In PF, nibentan exhibited a concentration-dependent biphasic effect: the maximum prolongation was attained at 1 × 10⁷ M, and a decrease of APD₉₀ was then seen at higher concentrations (table 1; fig. 4A). This decrease was accompanied by significant shortening of APD₅₀ (fig. 4B). The biphasic concentration-dependence of the APD was a result of the biphasic pattern of the concentration-dependence of the slope of phase 2, whereas a monotonic decrease in the slope of phase 3 was observed (table 1). In contrast to PF, in both types of ventricular myocardial preparations, nibentan induced a concentration-dependent increase in the APD that attained a steady-state level at 1 × 10⁶ M (tables 2 and 3; fig. 4). The lengthening of the APD was not associated with a slowing of the terminal phase of repolarization and was a result of practically equal increases of APD₅₀ and APD₉₀. In atrial muscle, the compound produced a monotonic increase of APD₉₀ through the highest concentration studied (table 4; fig. 4). There were no significant changes in APD₅₀, which suggests that the increase of APD₉₀ was due to prolongation of the terminal phase of repolarization.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effects of nibentan on PF, ventricular subendocardial and subepicardial muscle and atrial muscle APD at 90% (panel A) and 50% (panel B) of full repolarization at a CL of 500 ms. C, Control. *P < .05 vs. control (n = 8 for PF, n = 6 for all types of myocardial tissues).

A remarkable difference in the use-dependent effects of nibentan on the APD of PF and myocardial tissue was found, as well (fig. 5A). The ability of nibentan to prolong the APD of PF significantly diminished as the CL shortened, whereas in ventricular and atrial muscle, it showed no reverse use-dependence and lengthened the APD at low and high rates to a similar extent. The reverse use-dependent effect on PF was associated with an increase in the slope of phase 2: as the CL shortened, the relative effect on the slope of phase 2 significantly increased (fig. 5B). The relative decrease in the slope of phase 3 was about the same at all CL.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   A) Relationship between the increase in APD at 90% repolarization induced by nibentan (1 × 106 M) in PF, ventricular subendocardial and subepicardial muscle and atrial muscle and a CL of stimulation. *P < .05 vs. increase at CL of 2000 ms (n = 8 for PF, n = 6 for all types of myocardial tissues). B) Relative changes in slopes of phase 2 and phase 3 of PF action potentials induced by nibentan (1 × 106 M) at different CLs of stimulation. *P < .05 vs. change at CL of 2000 ms (n = 8).

For the sake of comparison, the effects of l-sotalol (5 × 10⁵ M) (selected for reasons described in "Discussion") were studied in PF and in endocardial preparations. Like nibentan, l-sotalol manifested prominent reverse use-dependence in the lengthening of PF APD (fig. 6A). In contrast to nibentan, l-sotalol showed reverse use-dependence in ventricular myocardium as well. The reverse use-dependent effect of l-sotalol was also associated with a use-dependent increase in the slope of phase 2 (fig. 6B). However, in comparison with nibentan, the curve was shifted in the negative direction such that l-sotalol never increased the slope of phase 2 above control values at any CL. A difference between the compounds' effects on the slope of phase 3 was also observed: unlike nibentan, the l-sotalol-induced relative slowing of phase 3 slope diminished as CL shortened (fig. 6B).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   A) Relationship between the increase in APD at 90% repolarization induced by l-sotalol in PF (n = 6) and in ventricular subendocardial muscles (n = 6) and a CL of stimulation. *P < .05 vs. increase at CL of 2000 ms. B) Relative changes in slopes of phase 2 and phase 3 of PF action potentials induced by l-sotalol at different CLs of stimulation. *P < .05 vs. change at CL of 2000 ms (n = 6).

The effects of nibentan on PF action potential parameters at different [K⁺]₀ are summarized in table 5. Nibentan had no effect on MDP at any [K⁺]₀. Its depressant effects on V_max were practically the same at all [K⁺]₀. Nibentan induced a significant increase in both APD and ERP and had no significant effects on ERP/APD₅₀ and ERP/APD₉₀ ratios at all [K⁺]₀.

View this table: [in this window] [in a new window]   TABLE 5 Effects of nibentan (5 × 107 M) on Pukinje fiber action potential characteristics at different [K±]0 (CL = 1000 ms)

The effects of nibentan (1 × 10⁸ through 1 × 10⁶ M) on normal automaticity in PF (n = 6) were studied. Control values of measured parameters were as follows: rate 19 ± 7 beats/min MDP 97 ± 1 V, phase 4 slope 5.2 ± 2.0 mV/s and activation voltage 86 ± 2 mV. None of these parameters was significantly altered by nibentan.

In the presence of isoproterenol alone, there was a significant, concentration-dependent increase in PF automaticity (control rate was 21 ± 8 beats/min, and that in isoproterenol 10⁶ M was 83 ± 17 (n = 6, P < .05)). Nibentan (5 × 10⁷ M) completely eliminated the effects of isoproterenol (control rate was 14 ± 6 beats/min, and that in isoproterenol 10⁶ M was 17 ± 7 (n = 6, P < .05).

Nibentan also suppressed slow-response action potentials of 6 PF driven at a CL of 2000 ms. The compound had no effect on the AP amplitude (control = 67 ± 2 mV) and induced a moderate hyperpolarization (from a control of 53 ± 1 mV to 55 ± 1 mV at 10⁷ M; P < .05). It prolonged APD₅₀ (control = 105 ± 8 ms, 10⁷ M = 128 ± 11 ms; P < .05) and APD₉₀ (control = 157 ± 8 ms, 10⁷ M = 189 ± 11 ms; P < .05) and inhibited upstroke velocity in a concentration-dependent fashion (control = 8.9 ± 0.9 V/s, 10⁷ M = 7.9 ± 0.7 V/s; P < .05).

	Discussion

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Our results show that nibentan has no effect on the MDP of any tissue studied, which suggests that the compound does not affect the inward rectifier current (I_K1) responsible for maintaining the resting potential (Giles and Imaizumi, 1988). That nibentan does not alter phase 1 repolarization of the PF action potential suggests that it does not affect the transient outward current (I_to). Nibentan moderately attenuates V_max in a concentration- and use-dependent fashion in all tissues studied, which indicates that the compound has modest "local anesthetic" properties.

The most significant effect of nibentan in all tissues is a lengthening of the APD. In PF, this is accompanied by prolongation of the ERP, such that the ERP/APD ratio is never diminished. However, the effect on APD is associated with a so-called reverse use-dependence. There is a caveat here, in that two methods for evaluating the degree of use-dependence have been reported in different studies; at any CL, the APD lengthening has been expressed either in absolute values or as percentage changes. The relative (percentage) changes depend on two factors: the drug-induced absolute change in APD and the normal frequency-dependence of APD (Cohen et al., 1986), which differs in various cardiac tissues. These factors can confound the comparison of reverse use-dependence in different tissues. In addition, because of the progressive shortening of APD (especially in PF) at faster rates of stimulation, estimation of use-dependence with both methods in the same tissue can give significantly different results. The use of absolute values seems to us more reasonable, if only because the effect of any drug in impeding impulse propagation in a reentrant circuit would depend on the absolute extent of APD and ERP prolongation to eliminate an excitable gap.

The reverse use-dependence of nibentan effects in PF may have a potential drawback: PF have longer action potentials than other cardiac tissues, and their particularly great prolongation at slow rates may induce early afterdepolarizations, triggered activity and torsades de pointes arrhythmias (Brachman et al., 1983). However, there is a limit of nibentan-induced APD prolongation in PF: maximal prolongation is attained at 1 × 10⁷ M, and a shortening of APD occurs at higher concentrations. As a result, at the lowest stimulation rate (CL 2000 ms), the maximum increase in APD₉₀ induced by nibentan is 43% on average. This may be important with respect to the propensity of the compound to induce early afterdepolarizations. The relative shortening of the plateau (increase of the slope of phase 2) at high nibentan concentrations should make delayed afterdepolarizations less likely to occur.

In atrial and ventricular muscles, nibentan shows no reverse use-dependence in APD prolongation. This may be a favorable difference from most other drugs that prolong repolarization by blocking I_K (Hondeghem and Snyders, 1990; Funck-Brentano, 1993; Katritsis and Camm, 1993; Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991). As regards proarrhythmic propensity, it is also important that with an increase in concentration, the nibentan-induced APD increase in ventricular muscle attains a steady state at 1 × 10⁶ M. Only in atrial tissue is there a monotonic concentration-dependent increase in APD through the highest concentration studied (5 × 10⁶ M). However, there are no changes in atrial APD at the plateau potential, which makes the development of early afterdepolarizations less likely.

The comparison of l-sotalol with nibentan suggests one more mechanism for a low proarrhythmic propensity of the latter. L-Sotalol was used for comparison because it is identical to D-sotalol with respect to APD prolongation and I_K inhibition and yet, like nibentan, exhibits prominent beta adrenoreceptor blockade (Carmeliet, 1985; Manley et al., 1985). In the range of normal heart rates (CL 750-1000 ms), the l-sotalol-induced gradient of repolarization between PF and myocardium is greater than that seen with nibentan. Thus nibentan does not enhance spatial inhomogeneity of repolarization to the same extent as l-sotalol, which may make it less likely to be proarrhythmic.

The slow-response action potential may be responsible for certain reentrant arrhythmias and is also characteristic of the normal atrioventricular node (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991). The effect of nibentan in prolonging the duration of this action potential would suggest an ability to increase atrioventricular nodal refractoriness as well as to prolong refractoriness in some reentrant loops. Concentration-dependent inhibition of the upstroke velocity of slow-response action potentials suggests that nibentan suppresses Ca⁺⁺ current. This effect can be responsible for an increase in the slope of phase 2 of PF action potentials (that steepens the plateau) at high nibentan concentrations.

Previous results (Rosenshtraukh et al., 1995) and the present data are consistent with nibentan prolonging APD via block of the delayed rectifier potassium current. In this respect, the action of nibentan to counteract the effect of isoproterenol, which itself can be antiarrhythmic, assumes further importance in that I_K can be enhanced by beta adrenergic stimulation (Yazawa and Kameyama, 1990; Sanguinetti et al., 1991). Beta antagonism mitigates against any shortening of repolarization and refractoriness induced by catecholamines.

The prominent reverse use-dependence in nibentan-induced APD prolongation in PF but not in ventricular myocardium is of interest. It may be explained by differences in the mechanisms that underlie the frequency-dependence of APD in these tissues.

In PF, the major current responsible for repolarization is the delayed rectifier (I_K) (Cohen et al., 1986). Time constants of activation and inactivation of I_K are in the hundreds of milliseconds. It is generally thought that at fast stimulus rates, I_K does not deactivate completely during diastole and that the progressive accumulation of residual I_K is one of the mechanisms that produces the dramatic frequency-dependence of cardiac APD (Hauswirth et al., 1972; Cohen et al., 1986; Carmeliet, 1993). The frequency-dependent shortening of APD is accompanied by a significant acceleration of the slope of phase 3, consistent with the contributions of I_K to the final phase of repolarization in PF. We found that the nibentan-induced relative slowing of the slope of PF phase 3 was the same (~40%) at all CL, which suggests that the compound inhibits I_K independently of rate. It is important to emphasize here that even if a compound inhibits I_K independently of rate, it will display a reverse rate-dependence in APD prolongation. The reason for this can be seen in figure 7: the same relative slowing of the slope of phase 3 induces less APD prolongation when phase 3 is steep (at a high rate) than when it is gradual (at a slow rate). Thus in PF, reverse use-dependence can be a result (at least partly) of a normally existing frequency-dependence of the slope of phase 3. For nibentan, an increase in the slope of phase 2 makes an additional contribution to the reverse use-dependence of APD prolongation. This effect, which steepens the plateau, may be related to suppression of Ca⁺⁺ current and probably to a decrease in Na⁺ "window" current (Coraboeuf et al., 1979). Nibentan-induced acceleration of phase 2 occurs in a use-dependent fashion, and as a result, the compound steepens the plateau (i.e., shortens action potential) more at a high rate than at a low rate of stimulation.

View larger version (8K): [in this window] [in a new window]   Fig. 7.   The dependence of drug-induced APD prolongation on the slope of phase 3. The last portions of phase 2 and phase 3 of PF action potentials are schematically shown. For simplicity, phase 3 is presented as a straight line going from 0 to 90 mV. In panel A, the control slope of phase 3 equals 900 mV/s; in panel B, it is 450 mV/s. Drug-induced 50% slowing of phase 3 leads to APD prolongation of 100 ms when phase 3 is steep (panel A) and of 200 ms when phase 3 is gradual (panel B).

In contrast to PF, the shortening of APD in ventricular muscle is not accompanied by any significant changes in maximum rate of phase 3 repolarization, which suggests that the role of I_K in the final phase of repolarization is not so important in this tissue as in PF. In ventricular myocardium, the background inwardly rectifying potassium current (I_K1) makes a more important contribution to the final phase of repolarization than in Purkinje fibers (Shimoni et al., 1992). Because nibentan has no significant effects on I_K1 (it induces no changes in MDP in any cardiac tissue), it insignificantly (3%-10% at 1 × 10⁶ M) inhibits the slope of the final phase of repolarization. At the same time, rate-independent suppression of I_K by nibentan induces the rate-independent APD prolongation at the plateau level (the same lengthening of APD₅₀ and APD₉₀).

Atrial tissue exhibits much less frequency-dependence of APD than Purkinje or ventricular tissues. In atrium, l_to is much larger and I_K1 much lower than in ventricle (Hume and Uehara, 1985; Giles and Imaizumi, 1988), so the action potential in atrial cells has a short plateau and I_K plays a more prominent role in the final phase of repolarization. Nibentan appears to have no effect on I_to and to rate-independently inhibit I_K. This may explain why it induces approximately the same prolongation of atrial APD₉₀ at all CL and has no significant effect on APD₅₀.

The results with l-sotalol support our suggestions about the mechanism for the difference between nibentan effects on PF and myocardium. At concentrations used in the present study, sotalol has no effect on any inward ionic current and significantly inhibits I_K (Carmeliet, 1985). As a result, the compound decreases the slopes of phase 2 and 3 in PF. In contrast to nibentan, the effect of l-sotalol on the slope of phase 3 decreases with the increase in rate, a result consistent with a reverse use-dependence in I_K inhibition. These data could explain the difference between the effects of nibentan and l-sotalol in myocardial tissue, with a reverse use-dependent suppression of I_K by l-sotalol leading to reverse use-dependence in the lengthening of myocardial action potentials.

In conclusion, practically all compounds that inhibit I_K manifest some reverse use-dependence in APD prolongation that can depend on type of cardiac tissue and species as well. The results of the present study do not explain why nibentan inhibits I_K rate-independently, whereas l-sotalol is reverse use-dependent. Various mechanisms can account for this, such as the time constants of binding and unbinding of compounds from K channels (Carmeliet, 1993) or the ability of compounds to inhibit the slow (I_Ks) or the rapid (I_Kr) components of I_K (Sanguinetti and Jurkiewizc, 1990; Jurkiewizc and Sanguinetti, 1993). Regardless of this, we have demonstrated that, at least to a certain extent, the difference in degree of reverse use-dependence in various cardiac tissues can result from differences in mechanisms of their frequency-dependence of APD. As a result, a compound may lengthen APD in one type of tissue and shorten APD in another (Yabek et al., 1987; Anyukhovsky and Rosen, 1994). Thus experimental protocols that incorporate diverse cardiac tissues are more likely to reflect drug actions accurately than are experiments that rely on one or two tissue types.