Br. J. Pharmacol. (1991), 104, 726-730
(D Macmillan Press Ltd, 1991
Effects of flecainide on isolated vascular smooth muscles of rat Francisco Perez-Vizcaino, Juan Duarte & 'Juan Tamargo Department of Pharmacology, School of Medicine, Universidad Complutense, 28040-Madrid, Spain 1 The inhibitory effects of flecainide were studied on contractile responses in rat isolated aortae and caudal artery and on spontaneous mechanical activity in portal vein segments. 2 In rat isolated aorta flecainide, 1O-6m-5 10-0M, inhibited in a dose-dependent manner the contractile responses induced by high K (80mM) and noradrenaline (NA, 10- M). These inhibitory actions were observed when flecainide was added before or after the induced contractions and were similar in aortae with or without endothelium. 3 Contractile responses induced by addition of Ca to Ca-free high-K solution were also dosedependently inhibited by flecainide (IC50 = 2.5 + 0.3 x 10 -M). Moreover, flecainide inhibited the contractile responses elicited by NA in rings incubated in Ca-free solution. 4 Flecainide also inhibited the spontaneous mechanical activity in portal vein segments (ICj0 6.5 x
=
+ 0.9
x
1O 5M).
5 Flecainide, 10-4M, inhibited 45Ca uptake stimulated by high K or NA without altering 45Ca uptake in resting aortae. 6 These results indicated that in rat isolated aortae, caudal arteries and portal veins, flecainide inhibited Ca entry through voltage-operated channels and NA-induced Ca uptake as well as Ca release from intracellular stores, thus decreasing the availability of intracellular free Ca required for vascular smooth muscle contraction. Keywords: Flecainide; aorta; 45Ca movements; rat tail artery; portal vein
Introduction Flecainide acetate is a class Ic antiarrhythmic drug effective both in experimental and clinical ventricular and supraventricular tachyarrhythmias (Anderson et al., 1981; Duff et al., 1981; Campbell, 1983a; Hellestrand et al., 1984; Holmes & Heel, 1985; Somberg & Tepper, 1986). In cardiac muscle fibres, flecainide exhibits both very slow onset kinetics and recovery from V.ax block (Campbell & Vaughan Williams, 1983; Campbell, 1983a,b; Delpon et al., 1991) due to its high affinity for the activated and inactivated states of the Na channels (Anno & Hondeghem, 1990). In addition, there is some experimental and clinical evidence suggesting that flecainide may exhibit weak calcium channel blocking properties on cardiac tissues which can play some role in its antiarrhythmic effects. In fact, flecainide prolonged the PR interval of the ECG (Somberg & Tepper, 1986), depressed cardiac contractility both in vitro (Schulze & Knops, 1982) and in vivo (Serruys et al., 1983; Josephson et al., 1984) and inhibited the slow action potentials elicited by isoprenaline in ventricular muscle fibres partially depolarized with high K (Schulze & Knops, 1982). However, and even when the cardiac and haemodynamic effects of flecainide are well established (Josephson et al., 1984; Holmes & Heel, 1985; Silke et al., 1986), little is known about its direct action on peripheral blood vessels and the calcium antagonistic properties of flecainide. Therefore, the present study was undertaken to examine the effects of flecainide on contractile responses and 45Ca uptake in rat isolated vessels.
Methods
Experimental procedure Sprague-Dawley rats (either sex, 250-350g) were killed by a blow on the head. Thoracic aorta, portal vein and central caudal artery were dissected and placed immediately in a modified Krebs-Henseleit solution (KS) of the following comAuthor for correspondence.
position (mM): NaCl 118, KCI 4.75, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.2 and glucose 11. After excess of fat and connective tissue were removed, the aortae and central caudal artery were cut into rings about 2-3mm in length. Only the proximal part (0-2cm) of the caudal artery was used in these experiments (Bao et al., 1990). Aortic and caudal rings were mounted under a tension of 1 and 0.5 g respectively, by two parallel L-shaped stainless-steel holders (0.8 and 0.25mm in diameter, respectively) inserted into the lumen. One holder was connected to a Grass FT03 force-displacement transducer while the other served as anchor (Tamargo & Tejerina, 1989; Bao et al., 1990). Vein segments (2mm in width and 15mm in length) were mounted vertically under 1 g of tension in organ baths containing 20ml of KS as previously described (Carron et al., 1991). The tissue bath was maintained at 34°C and bubbled with 95% 02:5% CO2 gas mixture. Contractile responses were recorded isometrically on a Grass polygraph (Tamargo & Tejerina, 1989). The presence of endothelium in the aorta was confirmed by the relaxant effect of acetylcholine 1o- 7M-10-5 M against the contraction evoked by 10- 7M phenylephrine. In some arteries the endothelium was mechanically removed by gently rubbing the lumen. Each preparation was allowed to equilibrate for at least 90min before experiments were started and during this period the incubation medium was changed every 20 min. After equilibration the following experiments were performed: (1) In some experiments aortic and caudal rings were exposed to single submaximal concentrations of KCl (80 mM) and noradrenaline (NA, 10 -M). An initial 15min control contraction was obtained in each experiment with the appropriate stimulatory agent and rings were then rested for a minimum of 30 min. Control contractile responses for each agonist were obtained at the beginning of the experiment until two successive responses were almost identical in height. This was followed by exposure to flecainide for a 30 min period before the addition of KCl or NA and during the 25 min reexposure to stimulatory agents. Only one agonist was used in each experiment. The results of these experiments are expressed as a percentage of the maximal control agonistinduced contractile responses. (2) To determine whether flecainide could relax an existing contraction, aortic rings were contracted by single submaximal concentrations of NA or
VASCULAR EFFECTS OF FLECAINIDE
KCl. When the contractile response to either agonist was maximum, flecainide was added in progressively increasing cumulative concentrations (106-10-4M) at 30min intervals. The results were expressed as a percentage of the maximal control agonist-induced responses. (3) In additional experiments dose-response curves to CaCl2 (0.5-5 mM) were obtained in aortic rings incubated in Ca-free KS containing 2mm EGTA for 2h and then in Ca-free high-K (80mM) solution for 5min before addition of the drugs (Carron et al., 1991). The results are expressed as a percentage of the maximal contractile response induced by 5 mm CaCl2 . (4) To study the effects of flecainide on a membrane store of Ca mobilized by NA, the experimental protocol employed was similar to that previously described (Ebeibge & Aloamaka, 1985; Carron et al., 1991). Aortic rings incubated in Ca-free KS were stimulated by successive additions of 10-5 M NA for 5min ('Ca-washing'). When the contractile response to NA was suppressed the rings were exposed for 5 min to 2.7 mm Ca KS ('Ca-loading') and then again exposed to Ca-washing KS. The above protocol was repeated in the presence of flecainide, added at the end of the 'Ca-loading' period. Results are expressed as the percentage of the first contraction of the Cawashing period in the presence of flecainide. Control experiments were performed with the same experimental four protocols in the absence of flecainide.
Rat isolated atria Right and left atria were removed and mounted vertically under 1 g of tension in 20 ml organ baths containing Tyrode solution of the following composition (mM): NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaHCO3 11.9, NaH2PO4 0.42, and glucose 5.5 bubbled with 95% 02 and 5% CO2 at 340C. Right atria were allowed to beat spontaneously and left atria were electrically driven at a basal rate of 1 Hz through bipolar platinum electrodes with rectangular pulses (1 ms duration, twice threshold strength) delivered from a Grass SD-9 stimulator. Rate and amplitude of contractions were measured isometrically by a force-displacement transducer and recorded on a Grass polygraph (Perez-Vizcaino et al., 1991). Resting tension was adjusted to 1 g and a 30 min equilibration period was allowed to elapse before control measurements were made.
Measurements of45Ca uptake
727
Drugs The following drugs were used: flecainide -acetate (Laboratorios Dr. Esteve S.A.) and noradrenaline -bitartrate (Sigma Chem. Co., London). Flecainide as a powder was initially dissolved in distilled deionized water and further dilutions were made in KS. Ascorbic acid (10-kM) was added to each solution of NA, made up freshly every day. Throughout the paper values are expressed as the mean + s.e.mean and statistical analysis was performed with Student's t test. The differences between control and experimental values were considered significant when P < 0.05. Dose-response slopes were analyzed to obtain the concentration of flecainide producing a 50% inhibition of the maximal contractile response (IC50) by linear regression analysis (method of least squares).
Results
Effects on re-sting tension and myogenic portal activity At concentrations between 10-6m and S x 1i-oM, flecainide had no effect on base-line tension in aortic and caudal rings. In 6 portal vein segments the control amplitude of spontaneous contractions was 465.9 + 70.2mg. At 10- 5M, flecainide decreased the rate but slightly increased the amplitude of contractions (by 17.9 + 7.4%, P > 0.05). However, at higher concentrations flecainide inhibited in a concentration-dependent manner the amplitude of the contractions resulting in a complete suppression at 2 x 10-4M (IC50 = 6.5 + 0.9 x 10-5M). After a washout period of 40-60min the effects on the frequency and amplitude of contractions were almost completely reversed.
Effect on contractions induced by KCI and noradrenaline in aorta and tail artery The inhibitory effect of flecainide on contractile responses induced by KCl (80 mM) and NA (10- 5M) in rat aorta are shown in Figure la. Both agonists produced submaximal contractile responses of 687.6 + 91.7mg and 939.9 + 191.0, respectively (n = 8), these values representing 94.1 + 1.4% and 96.4 + 3.4% of the maximal contraction evoked by KCI 100mm and NA 1i-oM, respectively. At concentrations > 10- 5 M, flecainide produced a concentrationdependent inhibition of the contractile responses induced by both stimulatory agents, the IC50 being 3.5 + 0.8 x 1O-SM
The 45Ca uptake was estimated by measurement of the changes in specific activity of the calcium fraction resistant to displacement by lanthanum as previously described by Godfraind (1983) and Barrigon & Tamargo (1986). Aortic rings were equilibrated in KS of the following composition (mM): b a NaCl 122, KCl 5, NaHCO3 25, CaCl2 1.25, MgSO4 1.2 and glucose 11.5. This solution was adjusted to pH 7.4 and gassed 100 100with 95% 02:5% C02. After equilibration, experimental rings were treated with flecainide for 30 min. Then both -a experimental and control rings were incubated for 5min in KS containing 45Ca (specific activity 1 pCiml-P; ICN Bio- 0Q 50medicals, California) either with or without flecainide and 0 50then for another 2 min in the same soluticin with or without the addition of 80mM KCJ or 10-5M NA. At this time rings were washed for 5min in 500ml of a lanthanum solution of the following composition (mM): NaCl 1224 KCl 5.9, MgCl2 1.25, glucose 11, LaCl3 50 and Tris maleate 15 (pH 6.8) at 00C. -4 -3 -5 -6 -5 -4 -6 -3 A high concentration of lanthanum has been shown to remove (M) log [Flecainidel the extracellular 45Ca, while low temperature -inhibits transmembrane 45Ca fluxes and thus preserves intracellular 45Ca Figure 1 Effect of flecainide, 10-6M-5 x 10-'M, on the contractile (Karaki & Weiss, 1979). After this,rings were removed, pressed responses induced by high K (S, 80mM) and noradrenaline (0. with a roller and weighed. The rings were then placed in scin10-sM) in rat aortae (a) and rat tail artery (b). Ordinate scale: relaxthe ml of Protosol tillation vials and 0.5 (Dupont) added; rings ation (% of control values). Abscissa scale: flecainide concentration were digested overnight at 60°C. Radioactivity was assayed in (M). Each point represents the mean + s.e.mean of 8 experiments with a liquid scintillation counter (LKB Wallac 1211 Rack) as pres.e.mean shown by vertical bars. The asterisks denote significant differences (**P < 0.01) from control values. viously described (Barrigon & Tamargo, 1986). C
728
F. PtREZ-VIZCAINO et al.
and 8.4 + 0.5 x 10- M, respectively, which indicated that flecainide was significantly more potent in inhibiting the contractile responses induced by high K than those induced by NA (P < 0.01). The inhibitory effects of flecainide were completely reversed by washing with drug-free KS. Similar results were obtained in aortic rings after removal of the endothelium. Cumulative increases in the concentration of flecainide (10-6M-104M) in aortic rings previously contracted with 80 mm K and NA 10- M resulted in a concentrationdependent relaxation, but again flecainide was more potent against the contraction elicited by high K than by NA. Thus, the IC50 to relax the KCI-induced contraction was 8.6 + 3.1 x 10-6m, whereas at 10-4M flecainide relaxed aortic rings previously contracted with NA by only 39.1 + 5.7% of its maximum. The relaxant effects of flecainide were not altered after removal of the endothelium. The contractile responses elicited by high K (80mM) and NA 10- M in the rat tail artery were 379.3 + 80.0 and 525.6 + 156.2 mg, respectively (n = 8). As shown in Figure lb, flecainide, 10- 6M-10 - M, inhibited in a concentrationdependent manner the contractile responses induced by both agonists. However, and in contrast to aortic rings, flecainide was almost equipotent on the contractions evoked by both agonists, the IC50 values being 5.3 + 1.4 x 10-5M and 6.5 + 1.8 x 10 5 M (P > 0.05).
Effects on Ca-induced contractions of aortae The inhibitory effects of flecainide on the Ca-dependent contractile responses are shown in Figure 2. Following incubation for 2 h in Ca-free KS (2 mm EGTA) and then 5 min in high-K solution, the addition of Ca to the bathing media (0.5-5 mM) caused a contraction of the aortic rings in a concentrationdependent manner, the maximum contractile response induced by 5mM CaCl2 being 1088.3 + 146.2mg (n = 6). Flecainide, 10-5M, 5 x 10-5M and 10-4M produced a progressive decrease in the amplitude of Ca-induced contractions of K-depolarized aortic rings and caused a progressive shift of Ca concentration-response curves downwards and to the right. Regression analysis of the inhibition yielded an IC50 of 2.5 + 0.3 x 10 5M. 100-
Effects on noradrenaline releasable stores In another group of experiments the effects of flecainide were studied on the contractile responses induced by NA in rings which were incubated in Ca-free KS. At this time, stimulation with 10- 5M NA resulted in a phasic contraction of an amplitude of 249.5 + 39.2mg (n = 8), presumed to reflect Ca mobilization from a membrane-bound store (Karaki et al., 1979). Flecainide, 10-5M and 10-0M, when applied after 'Caloading', inhibited by 26.4 + 7.3% (P < 0.05) and 56.1 + 4.9%, (P
(D Macmillan Press Ltd, 1991
Effects of flecainide on isolated vascular smooth muscles of rat Francisco Perez-Vizcaino, Juan Duarte & 'Juan Tamargo Department of Pharmacology, School of Medicine, Universidad Complutense, 28040-Madrid, Spain 1 The inhibitory effects of flecainide were studied on contractile responses in rat isolated aortae and caudal artery and on spontaneous mechanical activity in portal vein segments. 2 In rat isolated aorta flecainide, 1O-6m-5 10-0M, inhibited in a dose-dependent manner the contractile responses induced by high K (80mM) and noradrenaline (NA, 10- M). These inhibitory actions were observed when flecainide was added before or after the induced contractions and were similar in aortae with or without endothelium. 3 Contractile responses induced by addition of Ca to Ca-free high-K solution were also dosedependently inhibited by flecainide (IC50 = 2.5 + 0.3 x 10 -M). Moreover, flecainide inhibited the contractile responses elicited by NA in rings incubated in Ca-free solution. 4 Flecainide also inhibited the spontaneous mechanical activity in portal vein segments (ICj0 6.5 x
=
+ 0.9
x
1O 5M).
5 Flecainide, 10-4M, inhibited 45Ca uptake stimulated by high K or NA without altering 45Ca uptake in resting aortae. 6 These results indicated that in rat isolated aortae, caudal arteries and portal veins, flecainide inhibited Ca entry through voltage-operated channels and NA-induced Ca uptake as well as Ca release from intracellular stores, thus decreasing the availability of intracellular free Ca required for vascular smooth muscle contraction. Keywords: Flecainide; aorta; 45Ca movements; rat tail artery; portal vein
Introduction Flecainide acetate is a class Ic antiarrhythmic drug effective both in experimental and clinical ventricular and supraventricular tachyarrhythmias (Anderson et al., 1981; Duff et al., 1981; Campbell, 1983a; Hellestrand et al., 1984; Holmes & Heel, 1985; Somberg & Tepper, 1986). In cardiac muscle fibres, flecainide exhibits both very slow onset kinetics and recovery from V.ax block (Campbell & Vaughan Williams, 1983; Campbell, 1983a,b; Delpon et al., 1991) due to its high affinity for the activated and inactivated states of the Na channels (Anno & Hondeghem, 1990). In addition, there is some experimental and clinical evidence suggesting that flecainide may exhibit weak calcium channel blocking properties on cardiac tissues which can play some role in its antiarrhythmic effects. In fact, flecainide prolonged the PR interval of the ECG (Somberg & Tepper, 1986), depressed cardiac contractility both in vitro (Schulze & Knops, 1982) and in vivo (Serruys et al., 1983; Josephson et al., 1984) and inhibited the slow action potentials elicited by isoprenaline in ventricular muscle fibres partially depolarized with high K (Schulze & Knops, 1982). However, and even when the cardiac and haemodynamic effects of flecainide are well established (Josephson et al., 1984; Holmes & Heel, 1985; Silke et al., 1986), little is known about its direct action on peripheral blood vessels and the calcium antagonistic properties of flecainide. Therefore, the present study was undertaken to examine the effects of flecainide on contractile responses and 45Ca uptake in rat isolated vessels.
Methods
Experimental procedure Sprague-Dawley rats (either sex, 250-350g) were killed by a blow on the head. Thoracic aorta, portal vein and central caudal artery were dissected and placed immediately in a modified Krebs-Henseleit solution (KS) of the following comAuthor for correspondence.
position (mM): NaCl 118, KCI 4.75, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.2 and glucose 11. After excess of fat and connective tissue were removed, the aortae and central caudal artery were cut into rings about 2-3mm in length. Only the proximal part (0-2cm) of the caudal artery was used in these experiments (Bao et al., 1990). Aortic and caudal rings were mounted under a tension of 1 and 0.5 g respectively, by two parallel L-shaped stainless-steel holders (0.8 and 0.25mm in diameter, respectively) inserted into the lumen. One holder was connected to a Grass FT03 force-displacement transducer while the other served as anchor (Tamargo & Tejerina, 1989; Bao et al., 1990). Vein segments (2mm in width and 15mm in length) were mounted vertically under 1 g of tension in organ baths containing 20ml of KS as previously described (Carron et al., 1991). The tissue bath was maintained at 34°C and bubbled with 95% 02:5% CO2 gas mixture. Contractile responses were recorded isometrically on a Grass polygraph (Tamargo & Tejerina, 1989). The presence of endothelium in the aorta was confirmed by the relaxant effect of acetylcholine 1o- 7M-10-5 M against the contraction evoked by 10- 7M phenylephrine. In some arteries the endothelium was mechanically removed by gently rubbing the lumen. Each preparation was allowed to equilibrate for at least 90min before experiments were started and during this period the incubation medium was changed every 20 min. After equilibration the following experiments were performed: (1) In some experiments aortic and caudal rings were exposed to single submaximal concentrations of KCl (80 mM) and noradrenaline (NA, 10 -M). An initial 15min control contraction was obtained in each experiment with the appropriate stimulatory agent and rings were then rested for a minimum of 30 min. Control contractile responses for each agonist were obtained at the beginning of the experiment until two successive responses were almost identical in height. This was followed by exposure to flecainide for a 30 min period before the addition of KCl or NA and during the 25 min reexposure to stimulatory agents. Only one agonist was used in each experiment. The results of these experiments are expressed as a percentage of the maximal control agonistinduced contractile responses. (2) To determine whether flecainide could relax an existing contraction, aortic rings were contracted by single submaximal concentrations of NA or
VASCULAR EFFECTS OF FLECAINIDE
KCl. When the contractile response to either agonist was maximum, flecainide was added in progressively increasing cumulative concentrations (106-10-4M) at 30min intervals. The results were expressed as a percentage of the maximal control agonist-induced responses. (3) In additional experiments dose-response curves to CaCl2 (0.5-5 mM) were obtained in aortic rings incubated in Ca-free KS containing 2mm EGTA for 2h and then in Ca-free high-K (80mM) solution for 5min before addition of the drugs (Carron et al., 1991). The results are expressed as a percentage of the maximal contractile response induced by 5 mm CaCl2 . (4) To study the effects of flecainide on a membrane store of Ca mobilized by NA, the experimental protocol employed was similar to that previously described (Ebeibge & Aloamaka, 1985; Carron et al., 1991). Aortic rings incubated in Ca-free KS were stimulated by successive additions of 10-5 M NA for 5min ('Ca-washing'). When the contractile response to NA was suppressed the rings were exposed for 5 min to 2.7 mm Ca KS ('Ca-loading') and then again exposed to Ca-washing KS. The above protocol was repeated in the presence of flecainide, added at the end of the 'Ca-loading' period. Results are expressed as the percentage of the first contraction of the Cawashing period in the presence of flecainide. Control experiments were performed with the same experimental four protocols in the absence of flecainide.
Rat isolated atria Right and left atria were removed and mounted vertically under 1 g of tension in 20 ml organ baths containing Tyrode solution of the following composition (mM): NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaHCO3 11.9, NaH2PO4 0.42, and glucose 5.5 bubbled with 95% 02 and 5% CO2 at 340C. Right atria were allowed to beat spontaneously and left atria were electrically driven at a basal rate of 1 Hz through bipolar platinum electrodes with rectangular pulses (1 ms duration, twice threshold strength) delivered from a Grass SD-9 stimulator. Rate and amplitude of contractions were measured isometrically by a force-displacement transducer and recorded on a Grass polygraph (Perez-Vizcaino et al., 1991). Resting tension was adjusted to 1 g and a 30 min equilibration period was allowed to elapse before control measurements were made.
Measurements of45Ca uptake
727
Drugs The following drugs were used: flecainide -acetate (Laboratorios Dr. Esteve S.A.) and noradrenaline -bitartrate (Sigma Chem. Co., London). Flecainide as a powder was initially dissolved in distilled deionized water and further dilutions were made in KS. Ascorbic acid (10-kM) was added to each solution of NA, made up freshly every day. Throughout the paper values are expressed as the mean + s.e.mean and statistical analysis was performed with Student's t test. The differences between control and experimental values were considered significant when P < 0.05. Dose-response slopes were analyzed to obtain the concentration of flecainide producing a 50% inhibition of the maximal contractile response (IC50) by linear regression analysis (method of least squares).
Results
Effects on re-sting tension and myogenic portal activity At concentrations between 10-6m and S x 1i-oM, flecainide had no effect on base-line tension in aortic and caudal rings. In 6 portal vein segments the control amplitude of spontaneous contractions was 465.9 + 70.2mg. At 10- 5M, flecainide decreased the rate but slightly increased the amplitude of contractions (by 17.9 + 7.4%, P > 0.05). However, at higher concentrations flecainide inhibited in a concentration-dependent manner the amplitude of the contractions resulting in a complete suppression at 2 x 10-4M (IC50 = 6.5 + 0.9 x 10-5M). After a washout period of 40-60min the effects on the frequency and amplitude of contractions were almost completely reversed.
Effect on contractions induced by KCI and noradrenaline in aorta and tail artery The inhibitory effect of flecainide on contractile responses induced by KCl (80 mM) and NA (10- 5M) in rat aorta are shown in Figure la. Both agonists produced submaximal contractile responses of 687.6 + 91.7mg and 939.9 + 191.0, respectively (n = 8), these values representing 94.1 + 1.4% and 96.4 + 3.4% of the maximal contraction evoked by KCI 100mm and NA 1i-oM, respectively. At concentrations > 10- 5 M, flecainide produced a concentrationdependent inhibition of the contractile responses induced by both stimulatory agents, the IC50 being 3.5 + 0.8 x 1O-SM
The 45Ca uptake was estimated by measurement of the changes in specific activity of the calcium fraction resistant to displacement by lanthanum as previously described by Godfraind (1983) and Barrigon & Tamargo (1986). Aortic rings were equilibrated in KS of the following composition (mM): b a NaCl 122, KCl 5, NaHCO3 25, CaCl2 1.25, MgSO4 1.2 and glucose 11.5. This solution was adjusted to pH 7.4 and gassed 100 100with 95% 02:5% C02. After equilibration, experimental rings were treated with flecainide for 30 min. Then both -a experimental and control rings were incubated for 5min in KS containing 45Ca (specific activity 1 pCiml-P; ICN Bio- 0Q 50medicals, California) either with or without flecainide and 0 50then for another 2 min in the same soluticin with or without the addition of 80mM KCJ or 10-5M NA. At this time rings were washed for 5min in 500ml of a lanthanum solution of the following composition (mM): NaCl 1224 KCl 5.9, MgCl2 1.25, glucose 11, LaCl3 50 and Tris maleate 15 (pH 6.8) at 00C. -4 -3 -5 -6 -5 -4 -6 -3 A high concentration of lanthanum has been shown to remove (M) log [Flecainidel the extracellular 45Ca, while low temperature -inhibits transmembrane 45Ca fluxes and thus preserves intracellular 45Ca Figure 1 Effect of flecainide, 10-6M-5 x 10-'M, on the contractile (Karaki & Weiss, 1979). After this,rings were removed, pressed responses induced by high K (S, 80mM) and noradrenaline (0. with a roller and weighed. The rings were then placed in scin10-sM) in rat aortae (a) and rat tail artery (b). Ordinate scale: relaxthe ml of Protosol tillation vials and 0.5 (Dupont) added; rings ation (% of control values). Abscissa scale: flecainide concentration were digested overnight at 60°C. Radioactivity was assayed in (M). Each point represents the mean + s.e.mean of 8 experiments with a liquid scintillation counter (LKB Wallac 1211 Rack) as pres.e.mean shown by vertical bars. The asterisks denote significant differences (**P < 0.01) from control values. viously described (Barrigon & Tamargo, 1986). C
728
F. PtREZ-VIZCAINO et al.
and 8.4 + 0.5 x 10- M, respectively, which indicated that flecainide was significantly more potent in inhibiting the contractile responses induced by high K than those induced by NA (P < 0.01). The inhibitory effects of flecainide were completely reversed by washing with drug-free KS. Similar results were obtained in aortic rings after removal of the endothelium. Cumulative increases in the concentration of flecainide (10-6M-104M) in aortic rings previously contracted with 80 mm K and NA 10- M resulted in a concentrationdependent relaxation, but again flecainide was more potent against the contraction elicited by high K than by NA. Thus, the IC50 to relax the KCI-induced contraction was 8.6 + 3.1 x 10-6m, whereas at 10-4M flecainide relaxed aortic rings previously contracted with NA by only 39.1 + 5.7% of its maximum. The relaxant effects of flecainide were not altered after removal of the endothelium. The contractile responses elicited by high K (80mM) and NA 10- M in the rat tail artery were 379.3 + 80.0 and 525.6 + 156.2 mg, respectively (n = 8). As shown in Figure lb, flecainide, 10- 6M-10 - M, inhibited in a concentrationdependent manner the contractile responses induced by both agonists. However, and in contrast to aortic rings, flecainide was almost equipotent on the contractions evoked by both agonists, the IC50 values being 5.3 + 1.4 x 10-5M and 6.5 + 1.8 x 10 5 M (P > 0.05).
Effects on Ca-induced contractions of aortae The inhibitory effects of flecainide on the Ca-dependent contractile responses are shown in Figure 2. Following incubation for 2 h in Ca-free KS (2 mm EGTA) and then 5 min in high-K solution, the addition of Ca to the bathing media (0.5-5 mM) caused a contraction of the aortic rings in a concentrationdependent manner, the maximum contractile response induced by 5mM CaCl2 being 1088.3 + 146.2mg (n = 6). Flecainide, 10-5M, 5 x 10-5M and 10-4M produced a progressive decrease in the amplitude of Ca-induced contractions of K-depolarized aortic rings and caused a progressive shift of Ca concentration-response curves downwards and to the right. Regression analysis of the inhibition yielded an IC50 of 2.5 + 0.3 x 10 5M. 100-
Effects on noradrenaline releasable stores In another group of experiments the effects of flecainide were studied on the contractile responses induced by NA in rings which were incubated in Ca-free KS. At this time, stimulation with 10- 5M NA resulted in a phasic contraction of an amplitude of 249.5 + 39.2mg (n = 8), presumed to reflect Ca mobilization from a membrane-bound store (Karaki et al., 1979). Flecainide, 10-5M and 10-0M, when applied after 'Caloading', inhibited by 26.4 + 7.3% (P < 0.05) and 56.1 + 4.9%, (P
