Br. J. Pharmacol. (1993), 109, 873 879

Effects of lisinopril on electromechanical properties andmembrane currents in guinea-pig cardiac preparations

'Carmen Valenzuela, Onesima Perez, Oscar Casis, Juan Duarte, Francisco Perez-Vizcaino, EvaDelpon & Juan Tamargo

Department of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain

1 The effects of the angiotensin-converting enzyme inhibitor, lisinopril, were studied in guinea-pig atriaand papillary muscles and in single isolated ventricular cells.2 In isolated right atria, lisinopril (0.001 -10IOM) decreased the amplitude and rate of the spontaneouscontractions. In electrically driven left atria this negative inotropic effect was accompanied by ashortening of the time to peak tension and time for total contraction.3 Lisinopril did not modify the electrophysiological characteristics of the ventricular action potentialsrecorded in papillary muscles perfused with normal Tyrode solution or elicited by isoprenaline inpapillary muscles perfused with 27 mM K Tyrode solution.4 In single ventricular cells, lisinopril (10 pM) had no effect on the inward L-type Ca2" (kCaL), theinward rectifier (IKI) or the delayed rectifier K+ currents (IK). However, it abolished the stimulation-dependent facilitation of the L-type Ca2+ current.6 These results indicate that the negative inotropic effect of lisinopril cannot be explained by a decreasein Ca2+ entry through L-type channels and suggest that lisinopril may possibly act at an intracellularsite to reduce contractile force.

Keywords: Lisinopril; atrial and ventricular muscle; action potentials, L-type Ca2+ current; K+ currents; ventricular cells ofguinea-pig

Introduction Methods

Since their introduction, the angiotensin-converting enzymeinhibitors have an established role in the treatment ofhypertension and congestive heart failure (Braunwald, 1991).Moreover, recent evidence confirmed that chronic angio-tensin-converting enzyme inhibition may improve survival inexperimental models (Pfeffer et al., 1985; Sweet et al., 1987)and in patients with asymptomatic (SOLVD, 1992) andsevere heart failure of various aetiologies (Consensus, 1987;SOLVD, 1991; Pfeffer et al., 1992). Very recently, it has beenfound that captopril, a sulphydryl angiotensin-convertingenzyme inhibitor, reduced contractile force in guinea-pigisolated hearts (Walker et al., 1988), an effect which inisolated ventricular cells has been attributed to an inhibitionof Ca2+ entry through voltage-dependent L-type channels,ICa,L (Bryant et al., 1991). However, it is unknown whetherthis negative inotropic effect is related or not to the chemicalstructure (-SH group) of captopril.

Lisinopril, is a non sulphydryl, long-acting angiotensin-converting enzyme inhibitor that is the lysine analogue ofenalaprilat, the active form of enalapril (Case, 1987; Lan-caster & Todd, 1988). In patients with heart failure, lisinoprilproduced a sustained haemodynamic response and it hasproved to be as effective and well-tolerated as enalapril orcaptopril (Bach & Zardine, 1992; Zannad et al., 1992;Moyses & Higgins, 1992). Thus, the present experiments wereundertaken to: (1) characterize the electromechanical effectsof lisinopril in guinea-pig atria and ventricular papillarymuscles and (2) determine the effects of this drug on kCa,L, thedelayed rectifier (IK) and the inward rectifier K+ currents(IK1), in single ventricular myocytes isolated from guinea-pighearts. This would lead to a further insight into the ionicmechanisms responsible for the electrophysiological effects oflisinopril. A preliminary account of some of the results ofthis study has already been published (Valenzuela et al.,1992a).

' Author for correspondence.

Isolated atria

Guinea-pigs of either sex weighing 250-350 g were killed bycervical dislocation. The hearts were rapidly excised and rightand left atria were dissected and mounted vertically in a10 ml organ bath containing Tyrode solution of the followingcomposition (mM): NaCl 137, KCI 5.4, CaCl2 1.8, MgCl21.05, NaHCO3 24, NaH2PO4 0.42, glucose 5.5. The solutionswere bubbled with 95% 02 and 5% CO2 and maintained at34± 0.5°C. Under these conditions, right atria beat spon-taneously, while left atria were electrically driven at a basalrate of 1 Hz through bipolar platinum electrodes with rectan-gular pulses (1 ms duration, twice threshold strength)delivered from a programmable stimulator (Cibertec CS 220).Rate and amplitude of contractions were measured isomet-rically by FT03 force-displacement transducers and recordedon a Grass polygraph (Tamargo, 1980; Diez et al., 1985).Resting tension was adjusted to 1 g and a 30 min equilibra-tion period was allowed to elapse before controlmeasurements were taken. Peak contractile force, maximumrate of rise of force (d f/d t,a), time to peak tension and timefor total contractions were obtained from isometric tracings,and averaged from these successive contractions, aspreviously described (Diez et al., 1985; Perez-Vizcaino et al.,1991).After control values for each parameter were obtained,

incremental concentrations of lisinopril were added to thebath, to obtain a complete concentration-response curve. Theinterval between drug concentrations was 30 min, sincepreliminary time-response studies indicated that at this timethe effects have stabilized. The values for the differentparameters, obtained in the absence of lisinopril, were usedas a control and compared with those obtained after eachincrement in drug concentration.

Electrophysiological recordings

The experiments were performed in papillary muscles<1 mm in diameter isolated from the right ventricle. The

Br. J. Pharmacol. (I 993), 109, 873 879 '." Macmillan Press Ltd, 1993

874 C.VALENZUELA et al.

muscles were pinned to the bottom of a Lucite chamber andsuperfused continuously at a constant rate of 7 ml min-'with Tyrode solution bubbled with 95% 02 and 5% CO2 andmaintained at 34 ± 0.5°C. The preparations were initiallydriven at 1 Hz and a period of 1 h was allowed for equilibra-tion during which a stable impalement was obtained. Elec-trical stimulation was applied to the surface of the prepara-tion through Teflon-coated bipolar electrodes of silver wire.Driving stimuli, were rectangular pulses (2 ms duration, twicethreshold strength) delivered to the preparation from a multi-purpose programmable stimulator (Cibertec CS-220).Transmembrane action potentials (AP) were conven-

tionally recorded through glass microelectrodes filled with3 M KCI (tip resistance of 8-15 MQ). The microelectrodewas connected via Ag-AgCl half-cell to a high impedance,capacity neutralizing amplifier (WPI model 701). The max-imum rate of depolarization (Vm..) of the action potentialwas obtained by electronic differentiation (Valenzuela et al.,1988; Delpon et al., 1989; 1991b). The differentiator used hadan upper limit of linearity of 1000 V s-' and possessedvariable input filters (3 Hz-260 Hz). Stimulus intensity andduration of the stimuli were adjusted to obtain a constantlatency (1-2 ms) between stimulus and upstroke of theaction potential to minimize latency-induced alterations inVma. (Valenzuela et al., 1988). Both action potential and Vmuxwere displayed on a storage oscilloscope (Tektronix 5104N)and the oscilloscope traces were photographed with a kymo-graphic camera (Grass C4). After a 1 h equilibration period,control measurements were performed. The preparationswere then superfused with Tyrode solutions containing in-creasing concentrations of lisinopril (0.01-1OI1M).

Slow responses were elicited in papillary muscles renderedinexcitable by depolarizing with 27 mM K Tyrode solution.Excitability, i.e. slow action potentials, was restored indepolarized muscles driven at 0.12 Hz by adding isoprenaline(1 gm) to the perfusate (Delp6n et al., 1989; 1992).

Isolation of cardiac myocytes

Ventricular myocytes were isolated by enzymatic dissociationfollowing the procedure previously described (Valenzuela &Bennett, 1991; Delpon et al., 1991a). Guinea-pigs of either sex(200-300 g) were killed by cervical dislocation. Their heartswere rapidly removed and mounted on a Langendorff per-fusion system for retrograde coronary perfusion at a constantrate of 10-15 ml min'. Hearts were initially perfused for1-2 min with a modified Tyrode solution which contained1.8 mM CaC12 and then with a nominally calcium-free solu-tion at 37°C for a further 5 min. Thereafter, the hearts wereperfused for another 4 min with the same solution sup-plemented with 0.12 mg ml-' collagenase (type la, SigmaChemical Co., London) and 0.03 mg ml- ' protease (typeXIV, Sigma Chemical Co., London). Afterwards the heartswere washed with a high-K+, low Cl- solution (KB solution)for 4 min and then removed from the Langendorff columnand the ventricles dissected and cut into small pieces whichwere placed in a beaker containing KB solution and gentlyshaken to disperse the isolated cells. The resulting cell pelletwas stored in KB medium for 1-2 h before beginning theexperiment. The composition of the Tyrode solution was(mM): NaCl 114, KCI 5.4, CaCl2 1.8, MgCl2 1.0, taurine 20,glucose 10, NaHCO3 24 and NaH2PO4 0.42; pH was adjustedto 7.4 by addition of NaOH. The solution was bubbled with95% 02:5% C02. Nominally calcium-free Tyrode solutionwas prepared by simply omitting CaCl2 from the Tyrodesolution. The KB solution contained (mM): glutamic acid 70,taurine 10, KCI 20, KH2PO4 10, MgCl2 1.0, succinic acid 5.0,creatine 5.7, dextrose 10.0, EGTA-K 0.2 and HEPES-K 10.0;the pH was adjusted to 7.4 by addition of KOH. Allexperiments were performed at room temperature (22-23°C)on calcium-tolerant healthy ventricular myocytes (80-100 Amin length) identified by their rod-like and uniformly spacedcross-striated appearance.

A small aliquot of a suspension of dissociated cells wastransferred to a 0.5 ml chamber placed on the stage on aninverted microscope (Nikon TMS, Nikon Co., Tokyo). Aftersettling to the bottom of the chamber, cells were superfusedwith Tyrode solution at 1 ml min-'.The recording pipette (internal) solution contained (mM):

K-aspartate 80, KCI 42, KH2PO4 10, phosphocreatine 3,HEPES 5.0, MgATP 4, EGTA 5 (pH 7.2 with KOH). Whenanalysing the K+ currents the external solution contained(mM): NaCl 136, KCI 5.4, CaCl2 1.0, MgCl2 1.0, CoCl2 2.0,tetrodotoxin (TTX) 0.03, glucose 10 and HEPES 10; the pHwas adjusted to 7.4 by addition of NaOH. To minimizeoutward K+ currents when analysing the ICaL, a rich Csinternal solution was used in which K-aspartate and KClwere replaced by Cs-aspartate and CsCl, respectively, and thepH was adjusted to 7.4 by CsOH.

Current recording

Membrane currents were measured by the perforated nystatinpatch configuration of the patch-clamp technique (Hamill etal., 1981; Horn & Marty, 1988) using an Axopatch-IC clampamplifier (Axon Instruments, Foster City, CA, U.S.A.). Thismethod led to permeabilizing cardiac myocytes and recordingcalcium and potassium currents without a washout of theintracellular media, thus preventing the 'run-down' whichthese currents exhibit with classical whole-cell recording(Kurachi et al., 1989; Valenzuela et al., 1992b). Micropipetteswere constructed from borosilicate glass capillary tubes(Narishige GD-1, Narishige Co., Ltd., Tokyo, Japan) using aprogrammable patch micropipette puller (Model P-87 Brown-Flaming, Sutter Instruments Co., Novato, CA, U.S.A.). Thepipettes had a resistance of 2-4 MQ when filled with theinternal solution and immersed in the Tyrode solution. Aliquid junction potential of - 9 mV between the pipette solu-tion and the external solution was corrected for. Series resis-tance was compensated by an analog circuit. Capacitativeand linear leak currents were subtracted by an analoguecircuit or digitally by computer. Voltage-clamp commandpulses were generated by a 12-bit digital-to-analogue con-verter. Membrane currents were filtered at 1 kHz (- 3 dB) bya four-pole Bessel filter before sampling was done at 2 kHzby a 12-bit analogue-to-digital converter. During theexperiments, membrane potential and current data were dis-played on a storage oscilloscope (Tektronix model 7854,Beaverton, OR, U.S.A.) and stored on the hard disk of aHewlett Packard Vectra QS/16S computer using ananalogue-to-digital converter (Interface TL-1, Axon Instrum-ents Inc., Burlingame, CA, U.S.A.) controlled by acquisitionand display software (pClamp 5.5. Axon Instruments Inc.)(Valenzuela & Bennett, 1991; Delpon et al., 1991a; Valen-zuela et al., 1992b).The ICa,L was elicited by applying depolarizing clamp steps

of 500 ms from a holding potential of - 40 mV at a rate of0.3 Hz. Peak inward ICa,L was measured relative to the zerocurrent level (bath ground). Steady-state membrane currentswere monitored by applying hyperpolarizing and depolarizingclamp steps of S s duration from a holding potential of- 40 mV at a rate of 0.05 Hz. The steady-state membranecurrent was measured as the net current at the end of the 5 sclamp step referred to the zero current level. The delayedrectifier K+ current (IK) was monitored by measuring theoutward tail currents elicited at the end of 5 s depolarizingclamp steps and return to - 30 mV. The amplitude of tailcurrents was measured as the difference between the peakoutward tail current and the steady current level after decayof the tail current. A holding potential of - 40 mV was usedto inactivate the INa and the T-type Ca2" channels.

After a 15 min period for control measurements, the per-fusate was changed to one containing 1OIM lisinopril anddata were collected again after O min.

CARDIAC ELECTROPHYSIOLOGICAL EFFECTS OF LISINOPRIL 875

Drugs

Lisinopril (ICI Pharma, S.A.) as a powder was initially dis-solved in distilled deionized water to make 1 mM stock solu-tions. Further dilutions were carried out in Tyrode solutionto obtain final concentrations between 0.01 and 10JM.

on the resting membrane potential, phase 0 characteristics(amplitude and Vma,,) and duration of the ventricular actionpotentials at 50% (APDm) and 90% (APD90) repolarizationat any of the concentrations tested. No changes were alsoobserved in the duration of the ventricular effective refrac-tory period.

Data analysis and curve fitting

All data are given as mean ± s.e.mean and Student's t testwas used to estimate the significance of differences fromcontrol values. To analyse block at different voltages or drugconcentrations, two-way analysis of variance and Scheffe'stest for critical difference were used (Wallenstein et al., 1980).A P value of less than 0.05 was considered to be significant.

IK activation curves were fitted by a Boltzmann distribu-tion using a least-squares fitting routine described by Mar-quardt (1963)

IK/IKmax = l/(1 + exp[(Vh-Vm)/k])where Vh is the half-point of activation (in mV), Vm is thetest potential and k the slope factor for the activation curve(in mV).

a

100 -

-3C 50-00.1

Results

Effects on atrial rate and contractile force

The effects of lisinopril, 0.001 -10 JIM were studied on rateand amplitude of spontaneous contractions in 9 right atria.Control values for rate and contractile force averaged160.0 ± 6.8 beats min-' and 510 ± 30.0 mg, respectively. Asis shown in Figure la, lisinopril (0.01-110 IM) produced aconcentration-dependent decrease in the amplitude of atrialcontractions which reached statistically significant values atconcentrations >, than 0.1 JIM. However, at the range ofconcentrations studied, lisinopril has no effect on atrial rateand sinus node recovery time (not shown).

In 9 left atria driven at a basal rate of 1 Hz the averagedcontrol values of the isometric contractile parameters were:peak contractile force = 631.5 ± 29.8 mg, d fld tmax = 14.3 +1.2 mg ms-', time to peak tension = 65.3 ± 3.9 ms and thetime for total contraction = 211.1 ± 9.1 ms. Figure lb showsthat lisinopril (0.01-1O IM) also decreased the peak contrac-tile force in a concentration-dependent manner, an effectwhich reached significant values at all concentrations tested.This negative inotropic effect was accompanied by a paralleldecrease in d f/d tmax and by a significant shortening of thetime to peak tension and time for total contraction.

Effects on action potential characteristics

The effects of lisinopril in a range of concentrations between0.01JIM and 1O JM were studied in 8 ventricular papillarymuscles stimulated at a basal frequency of 1 Hz. Resultsobtained under control conditions and 30 min after eachincrement in drug concentration are shown in Table 1. It canbe observed that lisinopril did not produce significant effects

0-

b100

4-6

c 50-0

0c J

C 0.01 0.1 1 10

I

C 0.01 0.1 1 10

[Lisinoprill (>LM)

Figure 1 (a) The effect of lisinopril on rate (0) and amplitude (0)of spontaneous contractions in guinea-pig right atria; (b) the effect oflisinopril on peak contractile force (0), maximum rate of rise offorce (0), time to peak tension (A) and time for total contraction(A) in left atria driven at a basal rate of 1 Hz. Ordinate scale:percentage of control values. Abscissa scale: lisinopril expressed asmicromolar concentration. Each point represents the mean of 8experiments ± s.e.mean.

Table 1 Electrophysiological effects of lisinopril on transmembrane action potentials in guinea-pig papillary muscles driven at 1 Hz

Drugconcentration(AM)

00.010.1110

RMP(mV)

82.0 ± 0.882.6 ± 0.782.0 ± 0.882.0 ± 0.782.0 ± 1.2

APA(mV)

118.0 ± 1.6117.0 ± 2.2117.6± 1.7117.6± 1.9117.8± 1.9

Vmax(V s-')

185.4 ± 8.3184.9 ± 8.2183.6 ± 7.8189.0 ± 8.7194.6 ± 8.6

APD_s(ms)

161.3 ± 10.9148.2 ± 10.6150.3 ± 8.7145.3 ± 10.5141.4± 11.1

APD9o(ms)

191.3 ± 10.9180.4± 10.8181.9 ± 10.2177.6± 11.0171.8± 11.6

Values are given ± s.e.mean. n = 8.

876 C.VALENZUELA et al.

The effects of lisinopril were also studied on the charac-teristics of the slow action potentials induced by 1 JLMisoprenaline in papillary muscles perfused with 27 mM KTyrode solution and driven at a basal rate of 0.12 Hz. Table2 summarizes the results obtained in 8 muscles. Lisinoprilover the range of concentrations tested (0.01-10 AM) did notmodify the resting membrane potential, amplitude, Vm, andAPD50 and APD90 values of the slow action potentials. Theseresults suggest that the drug did not exhibit class IV antiar-rhythmic (Ca2l channel blocking) actions.

Effect on ICaL

To determine the effects of lisinopril (10JM) on the L-typeCa2+ current (VCaL), ventricular cells were clamped to a hol-ding potential of - 40 mV, and 500 ms depolarizing pulseswere applied every 40 s to membrane potentials between- 30 and + 70 mV. Figure 2a shows typical recording ofICa.L elicited when 500 ms depolarizing pulses were appliedfrom -40 mV to + 10 mV in control conditions and in thepresence of 10 JlM lisinopril. Under control conditions,depolarizing pulses were applied first, evoking the activationof an inward current, which was then followed by the inac-tivation process. As is shown, 10 LM lisinopril did not modifythe maximum amplitude or the inactivation time course ofthe ICa,L.

Figure 2b shows the current-voltage relationship when afamily of 500 ms depolarizing pulses were applied from - 40to membrane potentials between - 30 and + 70 mV in theabsence and in the presence of 10 JAM lisinopril. In theabsence of lisinopril, the current-voltage relation started at athreshold of - 30 mV, reached a maximum at + 10 mV andthe apparent reversal potential at + 50 mV. In 6 cells theaveraged peak amplitude of ICa,L at + 1OmV was 193.9 +35.0 pA. This current was abolished within 2-3 min afterperfusion with 2 mM Co-containing solution, an inorganicICa,L blocker (Isenberg & Klockner, 1982), but it was insen-sitive to 10 JuM tetrodotoxin (TTX) a specific blocker of INa.Therefore, the recorded inward current was identified as theICa,L. Lisinopril did not modify either the threshold potential,the potential at which the current reached its maximum or itsapparent reversal potential. These findings indicate thatlisinopril does not inhibit the ICa,L*

Effects of lisinopril on facilitation of ICa,L

An increase (facilitation) of L-type Ca2` current has beenobserved when increasing the rate of repetitive depolarizationin guinea-pig ventricular myocytes (Mitra & Morad, 1986;Lee, 1987; Zygmunt & Maylie, 1990). Figure 3 shows theeffect of stimulation upon ICaL. Under control conditions,following the application of a train of 16 depolarizing pulsesof 200 ms duration from a holding potential of - 40 mV to+ 1O mV, at a rate of 0.5 Hz, there was a progressive in-crease in the 'Ca,L amplitude, which is maximal within fivepulses. In 5 cells, the facilitation of the ICa,L measured as theincrease in the amplitude of the current, at the fifth pulse of

the train, averaged 20.1 ± 11.1%. The figure also shows thatthe initial rate of increase of the current was unchanged byrepetitive depolarization and that the facilitation of ICaL wasassociated with a reduction in the rate of inactivation. How-ever, in the presence of 10AM lisinopril this phenomenomwas abolished (- 0.05 ± 0.005% with respect to the firstpulse of the train; P<0.001).

a+10 mV

-40 mV

0 pA

Lis 10 pM

00

looms

b

801 il I

Vm (mV)

+70 mV 500ms

-40 mV-400-J

Figure 2 Effect of 10 lM lisinopril on L-type Ca2" current (ICa,L):(a) examples of current records after depolarizing clamp pulses of500 ms duration to +10 mV from a holding potential of - 40 mVbefore (Control) and after 10 min exposure to lisinopril (Lis); (b)effects of lisinopril on the current-voltage relationship of peak ICa,Lwhen applying 500 ms depolarizing pulses from - 40 mV to+70 mV in 1OmV steps as shown in the inset. Each point representsthe mean value obtained in 6 cells before (a) and after exposure tolisinopril (0). Standard error bars were omitted for clarity.

Table 2 Electrophysiological effects of lisinopril on slow actiondriven at 0.1 Hz

potentials induced by isoprenaline in guinea-pig papillary muscles

Drugconcentration(fAM)

00.010.1110

RMP(mV)

45.4± 1.645.0 ± 2.545.2± 1.545.6± 1.746.0 ± 2.0

APA(mV)

83.8 ± 1.083.0 ± 1.284.3 ± 1.282.4 ± 1.683.2 ± 0.8

Vmax(V s-')

14.8 ± 0.314.6 ± 0.114.6 ± 0.414.3 ± 0.214.3 ± 0.3

APD50(ms)

220.0± 10.8215.8 ± 12.3230.5 ± 12.4211.0± 17.6226.5 ± 10.3

APD90(Ms)

240.0 ± 10.9236.6 ± 14.4248.5 ± 13.4231.5 ± 18.9245.5 ± 10.5

Values are given ± s.e.mean. n = 8.

CARDIAC ELECTROPHYSIOLOGICAL EFFECTS OF LISINOPRIL 877

a Control Lisinopril (10 FJM)-120 ,,.

- 5th pulse

100 ms/m (nA) -4

-0.5-80

Vm (mV)

~~5 s+70 mV 5

- -40mV | -30mV

- -120 mV

b

150-

5100-

-u

50-

+10 mV _jj200 msjv

-40 mV-J -

*i*0 . .

-

Figure 4 Effects of lisinopril on the mean steady-state relationshipmeasured in 6 cells when applying 5 s depolarizing or hyperpolariz-ing pulses in 10 mV steps from a holding potential of -40 mV.Values were obtained during control perfusion (m) and following0 min exposure to 1OIM lisinopril (0). Cells were superfused with2 mm-' Co containing solution.

1.0 -

0.8 -

b pu

2 4 6 8 10 12 14 16Number of pulses

Figure 3 Effects of 10 JAM lisinopril on facilitation of ICa,L. The cellswere repetitively depolarized from a holding potential of - 40 mV toa test potential of + 10mV with a train of 16 pulses of 200 msduration at a frequency of 0.5 Hz. In (a), current traces obtainedduring the first (labelled 1) and fifth (labelled 5) pulse are superim-posed before (Control) and after 10 min exposure to lisinopril; (b)the facilitation of ICa,L during the application of a train of pulses in 5cells in the absence (L) and presence (0) of lisinopril. Standarderror bars were omitted for clarity.

= 0.6m

0)C_

-O 0.4-

0.2

0.0*

Effect on outward K currents

The effect of lisinopril on the delayed outward (IK) and theinward rectifying potassium currents (IKI) were also studiedin 6 cells. In these experiments the holding potential wasmaintained at - 40 mV to inactivate inward Na+ and T-typeCa2" currents. In addition, the external solution contained30 mM TTX and 2 mM CoC12 to block the Na+ and L-typeCa2+ currents, respectively. IK was activated by 5 s depolariz-ing pulses to potentials from - 30 to + 70 mV (Matsuura etal., 1987) and IKI by hyperpolarizing pulses ranging from- 50 to - 120 mV. Figure 4 shows the effects of lisinopril onthe current-voltage relationship. It can be observed thatneither the current amplitude of the current induced bydepolarizing pulses (IK) nor the current evoked by hyper-polarizing pulses negative to - 40 mV (IKI) was affected by10 JiM lisinopril.The effects of 1O AM lisinopril on the voltage-dependence

of IK was assessed by measuring the amplitude of the tailcurrents (Iuil) observed on return to - 30 mV after 5 s-depolarizing pulses to different test potentials (from - 30 to+ 70 mV in 10 mV steps) applied every 30 s from a holdingpotential of - 40 mV. In Figure 5, the currents were nor-malized relative to the maximum control Itajl amplitude foreach experiment (value of 1.0) and the resulting values wereplotted as a function of membrane potential. Under controlconditions the amplitude of I,l recorded at - 30 mV after adepolarizing pulse to + 50 mV averaged 149.4 ± 7.7 pA(n = 5). Lisinopril, 10 JAM, did not modify the maximum am-plitude of the I,, measured at - 30 mV (140.2 ± 10.5 pA,n = 5, P> 0.05). Under these experimental conditions, IK was

-30 -10 10 30 50Membrane potential (mV)

70

Figure 5 Effects of lisinopril on the voltage-dependance of IK wasassessed in 5 cells by measuring the amplitude of the tail currents(Iw,l) elicited on return to - 30 mV after 5 s depolarizing pulses froma holding potential of - 40 mV to different test potentials (from- 30 to + 70 mV in 10 mV steps) applied every 30 s. Values wereobtained during control perfusion (0) and following 10 minexposure to 1OJM lisinopril (0). The continuous line is the best fitobtained using a Boltzmann equation: IK/IKmax= 1/(1 +exp[(Vh-Vm)/k]), where Vh is the half-point of activation (in mV),Vm is the test potential and k the slope factor for the activation curve(in mV).

activated at potentials positive to - 30 mV and its amplitudeincreased at more depolarized levels. In the absence oflisinopril, the half-point (Vh) and the slope factor (k) of thesteady-state IK activation curves were 22.3 ± 2.4 mV and14.2 ± 0.2 mV (n = 6) and in the presence of lisinopril21.5 ± 2.9 mV (P>0.05) and 14.6 ± 0.8 mV (P>0.05),respectively.

Discussion

The main findings of this study are two fold. First, lisinoprilproduced a concentration-dependent decrease in peak con-tractile force in guinea-pig isolated atria. Second, thisnegative inotropic effect was produced by a range of concen-trations of lisinopril that did not modify atrial rate, nor thecharacteristics of fast (Na+-dependent) and slow (Ca2+-

Ido _O Mk-"

u

c

878 C.VALENZUELA et al.

dependent) ventricular action potentials, nor the inward Ca2"current (ICa,L), the delayed rectifier (IK) and the inwardrectifier K+ currents (IKI).

Both in spontaneously beating right atria and in electric-ally driven left atria lisinopril produced a concentration-dependent decrease in atrial contractile force. This negativeinotropic effect was accompanied by a parallel decrease in thed f/d tmax and by a concentration-dependent shortening of thetime to peak tension and the time for total contraction.However, lisinopril had no effect on atrial rate and sinusnode recovery time, which suggested that it does not exert adirect inhibition of the sinus node function. Captopril alsoproduced a negative inotropic effect in guinea-pig isolatedhearts (Walker et al., 1988) and this effect was attributed toan inhibition of ICaL (Bryant et al., 1991). However, in thepresent experiments the decrease in contractile forceappeared at concentrations at which lisinopril had no effecton the duration of the cardiac action potentials. Conse-quently, it seemed unlikely that its negative inotropic effectwas related to a decrease of Ca2" entry through L-typechannels. Moreover, lisinopril had no effect on the amplitudeof Vmax of the slow upstroke action potentials induced inK+-depolarized ventricular fibres where the INa has beenvoltage-inactivated. Under these conditions, excitability (i.e.slow action potentials) was restored by isoprenaline whichincreases the open probability of the L-type Ca2" channels(Tsien et al., 1986). Since the Vmax of the slow action poten-tials can be considered as a valid index of the ICa.L (Malecotet al., 1988), these results suggest that lisinopril has noinhibitory effect on this ionic current. To determine whetherlisinopril directly inhibits the kCaL, its effects were analysedon single isolated guinea-pig ventricular cells. Under theseconditions a concentration of lisinopril (10 LM), whichreduced the developed contractile force by almost 50%, hadno effect on the amplitude or the voltage-dependent proper-ties of the ICa,L. Therefore, and in contrast to what has beenpreviously described with captopril (Bryant et al., 1991), thepresent experiments demonstrated that the negative inotropiceffect of lisinopril cannot be explained through a decrease inCa2" entry via L-type channels.Another possible explanation for the decrease in contrac-

tility is that lisinopril may possibly act at an intracellular siteto reduce contractile force. Two experimental observationssupport this hypothesis. First, the negative inotropic effectwas accompanied by a shortening in the time to peak tensionand time for total contraction. These results suggested thatlisinopril may decrease the release of Ca2+ from the sarco-plasmic reticulum and/or increase the re-uptake of Ca2" inthe sarcoplasmic reticulum, but this cannot be determined onthe basis of the present results. Second, lisinopril suppressedthe increase (facilitation) of the ICa,L observed duringrepetitive depolarization in guinea-pig ventricular myocytes(Mitra & Morad, 1986; Lee, 1987; Zygmunt & Maylie, 1990).Facilitation of ICa,L seems to be mediated by a transient risein Ca2" near to the inner pore of Ca2" channels, resulting ina reduction in the rate of Ca2+-dependent inactivation of ICa,Land facilitation of Ca2+ current during subsequent depolari-

zations (Zygmunt & Maylie, 1990). Facilitation is alsoabolished by high concentrations of isoprenaline or caffeine(Tseng, 1988; Zygmunt & Maylie, 1990) which suggest thatfacilitiation of ICaL reflects a change in Ca2" channel phos-phorylation (Fedida et al., 1988). Whether lisinopril exerts asimilar effect at these levels deserves further investigation.

Lisinopril had no effect on resting membrane potential,amplitude and Vmax of the action potentials recorded in papil-lary muscles perfused with Tyrode solution. The lack of effecton phase 0 characteristics strongly suggested that lisinoprildid not exert an inhibitory effect on the fast inward sodiumcurrent (VNa) or the late Na+ current flowing during theplateau in guinea-pig ventricular myocytes (Kiyosue & Arita,1989). In addition, it explains why lisinopril did not modifyintra-atrial and intraventricular excitability and conductionin experimental animals and in man (Lancaster & Todd,1988). In guinea-pig ventricular cells the repolarization pro-cess is mainly due to the interplay between inward (ICa,L andslowly inactivating or late Na+ current) and outward cur-rents (IK and IKI) (Carmeliet & Vereecke, 1979; Kiyosue &Arita, 1989). The present study demonstrated that lisinoprildid not modify any of these ionic currents underlying themaintenance of the action potential plateau, which can ex-plain why no effect was observed on the duration of theaction potentials recorded either in normally polarized or indepolarized papillary muscles. Furthermore, the lack of effecton ionic currents evoked by clamp pulses negative to-30 mV, which are mainly due to the IKI, can explain why itdid not modify the resting membrane potential in papillarymuscles.The possible clinical relevance of the present results in

patients with congestive heart failure remains to be assessed.The beat dependence of facilitation of ICa,L is similar to thatof the positive staircase (Lee, 1987) and the decay of theinotropic state following postextrasystolic potentiation (Morad& Goldman, 1973). Thus, this phenomenon may play aphysiological role in the generation of the force staircase ofthe heart (Lee, 1987; Zygmunt & Maylie, 1990). However,the net haemodynamic effects of lisinopril result from acomplex interplay among direct and indirect cardiac andvascular actions each having the potential to influence themajor determinants of cardiac performance (Case, 1987; Lan-caster & Todd, 1988). In vivo, its most marked effect is adecrease in peripheral vascular resistances which explains thedecrease in systemic blood pressure and improved left ventri-cular systolic performance due to afterload reduction. Thus,it is possible that the direct negative inotropic effect oflisinopril can be counterbalanced in vivo by its potent arterialvasodilator action which reduced left ventricular afterload,overriding the expected direct cardiodepressant effects of thedrug.

We thank Dr M. Martin (ICI Farma, S.A.) for the generous gift oflisinopril. Financial support was provided by a CICYT Grant (SAF-92-0157) and by ICI Farma, S.A.

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(Received January 27, 1993Accepted March 3, 1993)