Introduction

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Propafenone is a class I antiarrhythmic drug used in the prophylaxis and treatment of both supraventricular and ventricular tachyarrhythmias (Funck-Brentano et al., 1990; Kishore and Camm, 1995). Because propafenone binds to the activated state of the Na⁺ channel and dissociates very slowly from its receptor site, it has been classified as a class Ic antiarrhythmic agent (Tamargo et al., 1992). In addition, at therapeutic concentrations propafenone exhibits -adrenoceptor blocking properties and inhibits several cardiac ionic currents such as the L-type Ca²⁺ current (Delgado et al., 1993) and different K⁺ currents, including the transient outward, I_to, rapid and slow delayed rectifiers (I_Kr and I_Ks, respectively), ATP-sensitive I_KATP, and the inward rectifier I_K1 (Duan et al., 1993; Delpón et al., 1995; Christé et al., 1999; Seki et al., 1999). These actions may account for its effects on the repolarization phase of cardiac action potential (Tamargo, 1993).

In isolated rat aorta and porcine coronary arteries, propafenone inhibited the contractile responses and the ⁴⁵Ca influx induced by a high concentration of KCl (Carrón et al., 1991; Pérez-Vizcaíno et al., 1994; Cogolludo et al., 1998). These effects have been attributed to its ability to block L-type Ca²⁺ channels. Moreover, in a recent study in rat aorta, propafenone was more potent than four other class I antiarrhythmic drugs in its ability to inhibit the vasodilatation induced by the K_ATP channel opener levcromakalim (Cogolludo et al., 1998). These results suggested that propafenone may also inhibit K_ATP channels in vascular smooth muscle cells. In fact, propafenone blocked K_ATP channels in isolated cardiac myocytes (Christé et al., 1999). Unfortunately, the effects of propafenone in vascular smooth muscle cells K⁺ currents are presently unknown.

The activity of potassium channels determines membrane potential and therefore vascular tone (Edwards and Weston, 1995; Nelson and Quayle, 1995). In vascular smooth muscle cells different types of potassium channels have been identified, including voltage-dependent channels with fast activating and inactivating kinetics (K_A), delayed rectifiers (K_V), large conductance Ca²⁺-activated channels (BK_Ca), K_ATP channels, and inward rectifiers (K_IR) (Edwards and Weston, 1995; Nelson and Quayle, 1995). All these currents have been well characterized in smooth muscle cells from portal veins, a widely used vascular preparation (Beech and Bolton, 1989; Hume and Leblanc, 1989; Edwards et al., 1994). The present experiments were, therefore, designed to study the effects of propafenone on K⁺ currents in freshly isolated smooth muscle cells from rat portal veins and on the spontaneous myogenic contractility in whole tissues.

	Materials and Methods

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Isolation of Cells. Male Wistar rats (250-300 g) were maintained under controlled light and temperature conditions, with food and water provided ad libitum. All experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986. Rat portal veins were dissected into a nominally calcium-free physiological salt solution (Ca²⁺-free PSS; see "Drugs and Solutions"). The veins were carefully cleaned of surrounding fat and connective tissue under a dissecting microscope, opened along their longitudinal axis, and the endothelium was removed by gently scratching the intimal surface with a metal rod. Tissues were then placed in the "enzyme solution" originally described for the isolation of guinea pig bladder smooth muscle cells (Klöckner and Isenberg, 1985) and agitated for 20 min at 37°C. Afterward tissues were washed in Ca²⁺-free PSS and cut into small segments that were then triturated by using a wide-bore, smooth-tipped pipette in Kraftbrühe (KB medium; Klöckner and Isenberg, 1985). Cells were stored at 4°C in KB medium and used within 8 h of separation. Cells used for experiments were either relaxed or partially contracted and exhibited spindle-shaped morphology; round and fully contracted cells were discarded. All experiments were performed at room temperature (22-24°C).

Electrophysiological Studies. A few drops of cell suspension were placed in a small volume (0.5 ml) bath mounted on the stage of an inverted phase-contrast microscope (model TMS; Nikon, Tokyo, Japan), and the cells were allowed to settle for 15 min and then continuously perfused with a Ca²⁺-free PSS at approximately 1 ml min¹. Single cells were voltage-clamped and membrane currents were measured using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981) by using an Axopatch-200B patch-clamp amplifier (Axon Instruments, Burlingame, CA). Patch pipettes were constructed from borosilicate glass capillaries (GD-1; Narishige, Tokyo, Japan) by using a programmable horizontal puller (Sutter Instruments Co., San Rafael, CA) and heat-polished with a microforge (MF-83; Narishige). The pipettes had resistances of 2 to 4 M when filled with the internal solution and immersed in the external solution. Currents were sampled at 4 kHz, filtered at 2 kHz, and digitized with a Digidata 1200 analog-to-digital converter (Axon Instruments). Series resistance compensation (60-80%) was performed in all the experiments. No leakage correction was applied. Current- and voltage-clamp protocols and data acquisition and analysis were performed using a computer (IBM compatible) and pClamp software (version 8.0; Axon Instruments). Data were temporarily stored on the computer hard disk for later analysis. Current-voltage (I-V) relationships were obtained by measuring the current at the peak or at the end of the 500-ms duration test pulses applied at a frequency of 0.1 Hz. All the currents were normalized for cell capacitance and expressed in pA pF¹. Cell capacitance was determined by integration of the capacity transient.

Measurement of Contractility. The portal veins were suspended vertically by means of two stainless steel holders under 1-g tension in 10-ml organ baths filled with Krebs' solution. One holder served as anchor and the other was attached to an isometric force transducer (model PRE 206-4; Cibertec, Madrid, Spain). Contractile tension was recorded by a REGXPC computer program (Cibertec) (Cogolludo et al., 1998; Pérez-Vizcaíno et al., 1999). Each preparation was allowed to equilibrate for 60 min before initiation of experimental procedures, and during this period the incubation medium was replaced every 30 min. The effects of propafenone (1, 3, and 10 µM), iberiotoxin (0.1 µM), and 4-aminopyridine (0.3 and 1 mM) were studied on the spontaneous contractions of the rat portal vein. After 20-min exposure to these drugs or vehicle (control), cumulative concentration-response curves to the potassium channel opener levcromakalim (0.01-10 µM) were carried out. Results were expressed as a percentage of the values obtained before adding these drugs.

Drugs and Solutions. The enzyme solution comprised 130 mM KOH, 0.05 mM CaCl₂, 20 mM taurine, 5 mM pyruvate, 5 mM creatine, 10 mM HEPES, 1 mg ml¹ collagenase (type VIII), 0.2 mg ml¹ pronase, 1 mg ml¹ fatty acid-free albumin, pH adjusted to 7.4 with methanesulfonic acid. The KB medium comprised 85 mM KCl, 30 mM KH₂PO₄, 5 mM MgSO₄, 5 mM Na₂ATP, 5 mM K-pyruvate, 5 mM creatine, 20 mM taurine, 5 mM -OH-butyrate, 1 mg ml¹ fatty acid-free albumin, pH adjusted to 7.2 with KOH.

Statistics. Data are expressed as means ± S.E.M; n indicates the number of cells tested. All experiments were conducted in cells from at least four different animals. Statistical analysis was performed using Student's t test for paired or unpaired observations. Differences were considered statistically significant when P was less than 0.05.

	Results

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Characteristics of K⁺ Currents in Portal Vein Myocytes under Calcium-Free Conditions

In the present study, the average capacitance of the freshly isolated rat portal vein myocytes was 28.9 ± 0.6 pF (n = 55). When rat portal vein cells were voltage-clamped at 80 mV the application of 500-ms depolarizing pulses to test potentials from 80 to +50 mV induced a family of K⁺ currents (I_Ktotal) that were usually reproducible for at least 1 h (Fig. 1A). The I_Ktotal became activated at potentials around 40 mV and its density at +50 mV averaged 31.8 ± 1.7 pA pF¹ when measured at the end of the depolarizing step. In most of the cells studied, the very fast activating and inactivating current [I_K(A)] was usually masked by a more prominent delayed rectifier current [I_K(V)] with slower activation and inactivation characteristics. Therefore, as previously described (Edwards et al., 1994, 1996), I_Ktotal flowing at the end of each voltage step and elicited from a holding potential of 80 mV comprised I_K(V) and I_NI and displayed outward rectifying properties (Fig. 1A). However, when the cells were held at 10 mV I_K(A) and I_K(V) inactivated, and therefore only I_NI was recorded when stepping to test potentials from 80 to +50 mV in 10-mV increments (Fig. 1B). This figure also shows that the I-V relationship of I_NI, which crossed the abscissa near 40 mV, exhibited a less pronounced outward rectification and its magnitude at +50 mV was approximately one-seventh that of I_Ktotal. I_Ktotal was only slightly affected by iberiotoxin (0.1 µM) but markedly inhibited by 4-aminopyridine (1 mM) (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effects of propafenone on outward K+ currents recorded in rat portal vein cells under Ca2+-free conditions. Current traces are shown for a series of 500-ms depolarization pulses from 80 to +50 mV (in 10-mV increments) from a holding potential of 80 (A) or 10 mV (B). The dashed line represents the zero current level. Under control conditions a family of K+ currents (IKtotal), which mainly comprised IK(V), was elicited when stepping from 80 mV. At a holding potential of 10 mV, IK(V) inactivated leaving only low-amplitude, noninactivating currents (INI). Propafenone (10 µM) inhibited IKtotal at all test potentials, but this inhibition was less prominent at potentials positive to +30 mV when the currents became very noisy. In cells held at 10 mV propafenone induced a noisy, outwardly rectifying current. Both panels also show the current-voltage relationship of IKtotal and INI measured at the end of the pulse in the absence and the presence of propafenone. Data represent mean ± S.E.M. (n = 6).

Effects of Propafenone on K⁺ Currents under Calcium-Free Conditions

Effects of Propafenone on I_K(V) and I_BK(Ca). When the cells were voltage-clamped at 80 mV, propafenone (10 µM) markedly inhibited I_Ktotal, even when at potentials positive to +30 mV the current became very noisy (compare currents obtained in the absence and the presence of propafenone in Fig. 1A). Furthermore, the magnitude of the inhibition of I_Ktotal induced by propafenone was significantly less prominent at test potentials positive to +30 mV (76.0 ± 2.2% inhibition at +10 mV versus 39.5 ± 9.7% inhibition at +50 mV; n = 6; P < 0.05). To further analyze this phenomenon, cells were held at 10 mV to inactivate I_K(A) and I_K(V) and then subjected to test potentials from 80 to +50 mV (Fig. 1B). At this holding potential, propafenone had no effect on the currents elicited between 80 and +20 mV but at more positive potentials induced a noisy outward current, so that at +50 mV the current density was significantly increased from 5.1 ± 0.6 to 10.2 ± 1.3 pA pF¹.

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View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of propafenone on outward K+ currents in rat portal vein cells previously perfused with, and in the continuous presence of, iberiotoxin (ITX) under Ca2+-free conditions. Current traces are shown for 500-ms depolarization pulses from 80 to +50 mV in steps of 10 mV from a holding potential of 80 (A) or 10 mV (B). The dashed line represents the zero current level. The perfusion with ITX (0.1 µM) produced a small reduction of IKtotal and INI elicited when the cells were held at 80 and 10 mV, respectively. Subsequent perfusion with ITX and propafenone (10 µM) markedly inhibited IKtotal and the noisy, outwardly rectifying current was not observed. Both panels also show the current-voltage relationship of IKtotal and INI measured at the end of the pulse in the absence and the presence of ITX and ITX plus propafenone. Data represent mean ± S.E.M. (n = 5).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   A, tracings showing the inhibitory effect of propafenone (10 µM) on IK(V) obtained by digital subtraction of INI from IKtotal. Currents were elicited by 500-ms pulses from 80 to +50 mV. B, current-voltage relationship of IK(V) measured at the end of the pulse in the absence and the presence of propafenone. Data represent mean ± S.E.M. (n = 6). C, concentration-dependent inhibition of IKtotal by propafenone. Individual data points are mean ± S.E.M. (n = 5-7). D, voltage dependence for the effects of propafenone (10 µM) on IKtotal in the absence and the presence of iberiotoxin (ITX, 0.1 µM) and on IK(V) (obtained by digitally subtracting INI from IKtotal). The percentage of inhibition of IKtotal induced by propafenone was significantly smaller at potentials positive to +30 mV compared with that obtained at +10 mV. No significant voltage dependence was observed for the inhibition of IK(V) or IKtotal in the presence of ITX. Data represent mean ± S.E.M. (n = 5-6).

Effects of Propafenone on I_K(A). As indicated above, in most of the cells I_K(A) was masked by I_K(V). However, in 9% (n = 5/55) of the cells studied, the currents evoked on stepping from a holding potential of 80 mV presented a clear transient outward current [I_K(A)] with fast activation and inactivation kinetics followed by a sustained outward current with slow inactivation characteristics (Fig. 4A). These cells were more elongated than the rest, spindle-shaped, and presented a greater capacitance (42.5 ± 4.3 pF; n = 5; P < 0.001), reflecting their larger size. Figure 4A shows the K⁺ currents present in one of these cells in control conditions and after perfusing with iberiotoxin and with iberiotoxin plus propafenone. The propafenone-sensitive current at +50 mV is illustrated in Fig. 4B. The current-voltage relationships for the peak current amplitude under these circumstances are shown in Fig. 4C. Under control conditions the application of depolarizing pulse to +50 mV yielded values of 47.3 ± 7.6 and 29.2 ± 6.3 pA pF¹ measured at the peak and at the end of the pulse, respectively. When these cells were superfused with iberiotoxin (0.1 µM) only a slight reduction (P > 0.05) of the amplitude of the current measured at the peak and at the end of the pulse was observed (40.3 ± 6.7 and 23.1 ± 6.7 pA pF¹, respectively). However, the subsequent perfusion with propafenone (10 µM) markedly inhibited (P < 0.05) the current both at the peak (17.2 ± 2.6 pA pF¹) and at the end of the voltage step (7.4 ± 2.6 pA pF¹). The effects of propafenone were partially reversed after drug washout. Figure 4C shows the I-V relationships for the peak current recorded in the absence and in the presence of iberiotoxin, alone or plus propafenone. Whereas iberiotoxin had little or no effect, in its continuous presence, propafenone significantly inhibited the peak current amplitude recorded at potentials positive to 40 mV. Figure 4D shows the I-V relationships of I_K(A) obtained by subtracting the I_Ktotal recorded at the peak from that recorded at the end of the pulse in the absence (control) and in the presence of iberiotoxin and iberiotoxin plus propafenone. The inhibitory effect of propafenone on I_K(A) was voltage-independent (Fig. 4E).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Effects of propafenone on IK(A) in rat portal vein cells previously perfused with iberiotoxin under Ca2+-free conditions. A, tracings showing a clear transient outward current [IK(A)] with fast activation and inactivation kinetics followed by a sustained outward current with slow inactivation characteristics [IK(V)]. Iberiotoxin (ITX, 0.1 µM) produced a small reduction, whereas propafenone (10 µM) markedly inhibited the current measured both at the peak and at the end of the 500-ms depolarizing steps. The dashed line represents the zero current level. B, tracing showing the propafenone-sensitive current from A. C, current-voltage relationship for the current measured at the peak in the absence and the presence of iberiotoxin and iberiotoxin plus propafenone. D, current-voltage relationship for IK(A) obtained by subtracting the IKtotal measured at the peak from that obtained at the end of the pulse in the absence and the presence of iberiotoxin and iberiotoxin plus propafenone. E, voltage dependence for the effects of propafenone on IK(A). Data shown in B to D represent mean ± S.E.M. (n = 5).

Effects of Propafenone on I_K(ATP). When the rat portal vein cells were voltage-clamped at 10 mV under Ca²⁺-free conditions, exposure to the potassium channel opener levcromakalim (10 µM) induced a large increase in the noninactivating current component by inducing I_K(ATP) (from 1.3 ± 0.1 to 4.7 ± 0.9 pA pF¹; P < 0.05). This current was fully inhibited by subsequent addition of propafenone (10 µM; 1.8 ± 0.2 pA pF¹) (Fig. 5A) or glibenclamide (10 µM; data not shown). Figure 5B shows that propafenone inhibited levcromakalim-induced current at test potentials between 60 and +20 mV, whereas at more positive potentials the inhibition of I_K(ATP) was compensated by an increase in the noninactivating current (see the noisy voltage-dependent outward current recorded in the presence of propafenone). This additional current was inhibited by iberiotoxin (0.1 µM), suggesting that at more positive potentials the inhibition of I_K(ATP) was compensated by the activation of I_BK(Ca).

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effects of propafenone on IK(ATP) induced by levcromakalim in rat portal vein cells under Ca2+-free conditions. A, typical trace showing the increase in outward current induced by levcromakalim (10 µM) and its inhibition by propafenone (10 µM) in a cell clamped at 10 mV. The dashed line represents the zero current. B, current traces are shown for 500-ms depolarization pulses from 80 to +50 mV in steps of 10 mV when holding the membrane potential at 10 mV. Under control conditions only INI was present. Levcromakalim enhanced the noninactivated current component, which comprised INI plus IK(ATP). The addition of propafenone inhibited levcromakalim-induced IK(ATP) but at potentials positive to +20 mV this inhibition was compensated by the induction of a noisy, outwardly rectifying current, which was inhibited by iberiotoxin (ITX, 0.1 µM). C, current-voltage relationship for the current measured at the end of the pulse in the absence and the presence of levcromakalim and levcromakalim plus propafenone. D, current-voltage relationship for IK(ATP), obtained by subtracting, for each test potential, the current elicited in the presence of levcromakalim (INI plus IK(ATP)) from that obtained under control conditions (INI) in the absence and the presence of propafenone. E, voltage dependence for the effects of propafenone on IK(ATP). Data shown in C to E represent mean ± S.E.M. (n = 5).

Effects of Propafenone on Potassium Currents in Calcium-Containing Conditions

To further study the effects of propafenone on BK_Ca channels portal vein myocytes were bathed with Ca²⁺-PSS (1.8 mM CaCl₂) and EGTA was omitted in the pipette solution. Under these conditions, propafenone inhibited the total outward currents measured at the end of the pulse [now comprising both I_K(V) and I_BK(Ca)] and recorded on stepping from 80 mV to test potentials between 40 and +50 mV (Fig. 6A). At a holding potential of 10 mV, only the noisy, noninactivating, outwardly rectifying current, I_BK(Ca), was elicited on stepping to test potentials from 40 to + 50 mV (Fig. 6B). Under these conditions, brief spontaneous transient outward K⁺ currents (STOCs) were recorded in 50% of the cells (Fig. 6C). STOCs were never recorded in Ca²⁺-free conditions and were also inhibited by 0.1 µM iberiotoxin (data not shown). Exposure to 10 µM propafenone inhibited the noisy, outwardly rectifying current at all the test potentials (Fig. 6B) and immediately abolished any STOC present (Fig. 6C).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of propafenone on outward K+ currents recorded in rat portal vein cells bathed with Ca2+-PSS (1.8 mM CaCl2) and with EGTA omitted in the pipette solution. Current-voltage relationships of whole-cell currents obtained when cells were cells held at 80 (A) and 10 mV (B), respectively, in the absence and the presence of 10 µM propafenone. C, STOCs were evident in some cells when the holding potential was held at 10 mV. Propafenone (10 µM) inhibited the whole-cell outward currents (A and B) and suppressed the STOCs (C). STOCs were rapidly reappeared after washout of the drug. The dashed line represents the zero current level. Data represent mean ± S.E.M. (n = 5).

Contractility Studies

The spontaneous contractions of the rat portal veins used in the present study had an amplitude of 423 ± 24 mg, a frequency of 3.92 ± 0.19 min¹, and a duration of 7.48 ± 0.34 s (n = 46). Figure 7 shows three typical recordings of the spontaneous activity of the rat portal vein in the absence (control) and the presence of 4-aminopyridine, iberiotoxin, and propafenone. The exposure to the K_V channel inhibitor 4-aminopyridine (0.3 and 1 mM) and the BK_Ca channel inhibitor iberiotoxin (0.1 µM) for 20 min increased the amplitude of the spontaneous contractions (Fig. 7; Table 1). Iberiotoxin did not modify the duration or the frequency of the spontaneous contractions. At 0.3 mM, 4-aminopyridine prolonged the duration but did not change the frequency of these contractions, whereas at the highest concentration (1 mM) the marked prolongation of the duration was accompanied by a significant reduction in the frequency of the contractions (Table 1) and an increase of the basal tone (715 ± 15 mg; n = 4). The exposure to propafenone (1, 3, and 10 µM) increased the amplitude, prolonged the duration (disrupting the regular pattern), and reduced the frequency of the spontaneous contractions (Fig. 7; Table 1). However, at 10 µM, propafenone exhibited a biphasic behavior, characterized by an initial significant increase in the amplitude of the contractions (Fig. 7; Table 1), which after 2 to 4 min was followed by a progressive decrease, until a stable level was reached at values similar to the control conditions within 15 min. In the presence of iberiotoxin (0.1 µM), propafenone still elicited a biphasic increase in the amplitude of the contractions (140 ± 8 and 115 ± 7% at the peak and the steady state, respectively, of the values obtained before addition of propafenone; n = 4).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Original recordings of the effects of K+ channel inhibitors and propafenone on spontaneous contractions in rat portal veins. Each panel shows typical traces of about 1-min period in the absence and the presence of the drug. A, KV channels inhibitor 4-aminopyridine (0.3 mM) increased the amplitude and duration, and disrupted the regular pattern, of spontaneous contractions. In contrast, the BKCa channel inhibitor iberiotoxin (ITX, 0.1 µM) increased the amplitude but did not modify the duration or the regular pattern of the contractions (B). C, propafenone (10 µM) produced similar effects than 4-aminopyridine on mechanical activity in intact portal veins.

We also analyzed the effects of the potassium channel opener levcromakalim (0.01-10 µM) in the absence and in the presence of propafenone (Fig. 8). Levcromakalim caused a concentration-dependent inhibition of the amplitude of the spontaneous contractions (E_max = 94.5 ± 3.1%; EC₅₀ = 0.132 ± 0.008 µM; n = 4-6) and reduced their frequency and duration. Propafenone (1, 3, and 10 µM) reduced the maximal response (E_max = 85.2 ± 5.1, 64.3 ± 1.9, and 42.5 ± 4.3%, respectively; P < 0.01 for 3 and 10 µM) and shifted to the right the concentration-response curve to levcromakalim in a concentration-dependent manner (EC₅₀ = 0.195 ± 0.021, 0.215 ± 0.035, and 0.407 ± 0.048 µm, respectively; P < 0.05 for 10 µM) (Fig. 8).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Effects of propafenone on the reduction of the amplitude of the spontaneous contractions induced by levcromakalim (0.01-10 µM) in rat portal veins. Propafenone (1, 3, and 10 µM) reduced the maximal response and shifted to the right the concentration-response curve to levcromakalim in a concentration-dependent manner. Results are expressed as mean ± S.E.M. (n = 4-6).

	Discussion

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The present study is the first to characterize the effects of propafenone on K⁺ currents in vascular smooth muscle cells. Our results can be summarized as follows: 1) In freshly isolated smooth muscle cells from rat portal veins, propafenone inhibits I_K(V), I_K(A), and I_K(ATP) under essentially Ca²⁺-free conditions and I_BK(Ca) and STOCs under Ca²⁺-containing conditions. 2) In whole tissues propafenone increased the amplitude and duration of the spontaneous contractions and reduced the inhibitory effect of levcromakalim on their amplitude. These results in organ bath studies correlated with those obtained in isolated rat portal vein cells and provided further evidence that propafenone inhibits several K⁺ channels in vascular smooth muscle.

Effects of Propafenone on I_K(V), I_K(A), and I_BK(Ca). The K⁺ currents present in portal vein cells have been previously characterized (Beech and Bolton, 1989; Hume and Leblanc, 1989; Edwards et al., 1994, 1996). Under control Ca²⁺-free conditions I_Ktotal predominantly comprised I_K(V), whereas I_BK(Ca) was only evident when cells were perfused with Ca²⁺-PSS and EGTA omitted in the pipette.

Effects of Propafenone on I_K(ATP). We have demonstrated that several class I antiarrhythmic drugs, including propafenone, produced a noncompetitive antagonism of the relaxation induced by the potassium channel opener levcromakalim in isolated rat aorta (Cogolludo et al., 1998). In the present study, propafenone inhibited I_K(ATP) induced by levcromakalim in a voltage-independent manner. The ability of propafenone to inhibit both K_ATP and K_V channels in vascular smooth muscle has also been observed with several imidazoline- and guanidine-containing compounds (Ibbotson et al., 1993) and with other drugs chemically distinct from these such as phencyclidine, ciclacindol, and several cytochrome P450 inhibitors (Edwards et al., 1996).

Correlation of Effects of Propafenone on K⁺ Currents and Vascular Tone. The spontaneous contractions of rat portal vein are generated by multispike electrical complexes that at their peak shift the membrane potential up to 0 mV (Southerton et al., 1988). K⁺ permeability through K_V channels is responsible for determining the membrane potential and depolarization-dependent repolarization in portal veins (Southerton et al., 1988; Hume and Leblanc, 1989). Because K_V channels activate at 40 mV, their blockade would be expected to increase the magnitude and duration of the spontaneous contractions of rat portal veins. Propafenone inhibited I_K(V), increased the amplitude and the duration of the spontaneous contractions and disrupted their regular pattern, effects similar to those obtained with 4-aminopyridine and several inhibitors of I_K(V) (Ibbotson et al., 1993; Edwards et al., 1996). Iberiotoxin increased the amplitude but did not modify the duration or the frequency of the spontaneous contractions and did not change the increase in the amplitude induced by propafenone. It is unlikely that the stimulatory effect of propafenone resulted from the inhibition of I_K(ATP) because glibenclamide had no effect on spontaneous activity (data not shown). At 10 µM, propafenone produced an initial transient increase in the amplitude of the contractions, which after 2 to 4 min was followed by a reduction to a stable level still higher than that observed before the addition of the drug. The delayed relaxant effect of propafenone could be attributed to its ability to inhibit Ca²⁺ entry through L-type Ca²⁺ (Carrón et al., 1991; Pérez-Vizcaíno et al., 1994). Furthermore, propafenone inhibited the reduction of the amplitude of the spontaneous contractions induced by levcromakalim in a manner suggestive of a noncompetitive antagonism as previously described in rat aorta (Cogolludo et al., 1998). These results further confirmed the inhibitory effect of propafenone on K_ATP channels.

Potential Relevance of Present Results. The peak effective plasma concentrations of propafenone range between 3.1 and 18.1 ng ml¹ (0.9-5.3 µM), but because it is about 90% protein bound (Bryson et al., 1993) in vitro concentrations of 0.2 to 0.6 µM probably correspond in action to clinically effective free drug plasma concentrations. Thus, the blockade of I_K(V) and I_K(ATP) produced by propafenone is encountered at clinically relevant concentrations. However, we must be cautious in extrapolating our results to the in vivo situation. The inhibition of K⁺ channels is expected to produce membrane depolarization, activation of L-type Ca²⁺ channels, increased intracellular calcium concentration, and vasoconstriction (Nelson et al., 1995). However, oral administration of propafenone had no effect on arterial blood pressure (Funck-Brentano et al., 1990), whereas after the intravenous administration conflicting results have been reported (Bryson et al., 1993). Thus, even when in most studies propafenone produced a transient and slight fall in systolic blood pressure (Beck et al., 1978; Connolly et al., 1987; Musto et al., 1988; Touboul et al., 1988; Bianconi et al., 1989), an increase (Feld et al., 1987) or no change in this parameter has also been reported (Terrosu et al., 1986). The apparent discrepancy between our results and the clinical evidence and among clinical studies can be due to the fact that the vascular effects of propafenone are the final result of the inhibition of K⁺ and L-type Ca²⁺ channels, of Ca²⁺ release from intracellular stores, and of its direct cardiodepressant effects. L-type Ca²⁺ channel blockers decrease intracellular calcium concentration, Ca²⁺ sparks, BK_(Ca) activity, and STOCs and abolish the vascular effects of iberiotoxin (Nelson et al., 1995; Jaggar et al., 2000). Thus, it is possible that at certain concentrations the direct effects of propafenone on vascular K⁺ channels may be counteracted by the blockade of Ca²⁺ channels. Finally, propafenone exerts direct cardiodepressant effects that may produce a baroreflex increase in systemic vascular resistances in response to the decrease in cardiac output (Funck-Brentano et al., 1990; Bryson et al., 1993). Therefore, the possible clinical relevance of the present results remains to be determined.