Effects of various antianginal drugs on sodium influx in rat brain synaptosomes and in rat heart muscle cells in culture

European Journal of Pharmacology, 138 (1987) 1-8

1

Elsevier EJP 00761

Effects of various antianginal drugs on sodium influx in rat brain synaptosomes and in rat heart muscle cells in culture M i c h r l e G r i m a *, M a r i a n n e F r e y s s - B e g u i n 1, E l i s a b e t h M i l l a n v o y e - V a n Brussel 1, N i c o l e D e c k e r and Jean Schwartz Institut de Pharmacologie (UA 589 CNRS), Facult~ de M@decine, 11, rue Humann, 67000 Strasbourg, and i U 7 INSERM, Laboratoire de Pharmacologic, H~pital Necker, 161 rue de S@vres, 75015 Paris, France

Received 10 November 1986, revised MS received 12 February 1987, accepted 10 March 1987

This paper describes the inhibitory effects of several antianginal drugs on 22Na uptake of the fast Na ÷ channel in rat brain synaptosomes and in rat heart muscle cells in culture. Calcium antagonists like verapamil, flunarizine, perhexiline, two perhexiline derivatives IPS 629 and IPS 672, and fl-adrenoceptor antagonists like propranolol and practolol were tested. IPS 672 was the most active compound on synaptosomes and heart muscle cells (ICs0 = 2.0 x 10-6 and 2.4 × 10 -6 M respectively). The relative potencies of the Ca 2+ antagonists tested on heart muscle cells were found to be IPS 672 > IPS 629 > perhexiline > flunarizine > verapamil. Verapamil was 55 and 10 times less active than IPS 672 on synaptosomes and heart cells respectively. Propranolol had an inhibitory activity comparable to that of flunarizine and was 100 times more active than practolol. It can be concluded that several antianginal drugs seem to interfere with the Na + fast channel on rat brain and heart. 22Na uptake; Brain synaptosomes; Heart muscle cells; Antianginal drugs; Na + channel; (Rat)

1. Introduction Calcium antagonists are effective antianginal drugs, but differ as regards chemical structures, pharmacological effects and therapeutic activities. Spedding (1985) distinguished three classes of calcium antagonists: class I includes all the dihydropyridines while verapamil belongs to class II. Class III comprises the diphenyl-alkylamines, cinnarizine and flunarizine, and also drugs such as perhexiline. Verapamil is used in the treatment of angina, flunarizine has been described as a vasodilator (Holmes et al., 1984) while perhexiline has proved a very effective antianginal drug (Pepine et al., 1978) but these compounds are weaker as calcium antagonists than the dihydropyridine * To whom all correspondence should be addressed: Institut de Pharmacologie, 11, rue Humann, 67000 Strasbourg, France.

group and are less specific (Fleckenstein, 1985), so other mechanisms of action could be envisaged. The antianginal drugs also comprise the/3-adrenoceptor antagonists, e.g. propranolol, which has antiarrhythmic properties linked with an effect on the fast inward N a ÷ channel (Tarr et al., 1973). McNeal et al. (1985) have shown recently that some Ca 2+ antagonists belonging to class II, such as verapamil, or to class III, such as cinnarizine, flunarizine and prenylamine which have chemical structures comparable to that of perhexiline, inhibit the binding of [3H]batrachotoxinin A20ct benzoate in rat brain synaptosomes. The B-adrenoceptor antagonist, propranolol, also inhibits the binding of [3 H]batrachotoxinin A20a benzoate but is less potent than the class II or class III Ca 2÷ antagonists. The batrachotoxin binding site is linked with the fast N a ÷ channel. Grima et al. (1986) confirmed the interaction between the fast N a + channel and the antianginal drugs and showed

0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

that these drugs also inhibited the binding of [3H]batrachotoxinin A20a benzoate on rat heart membranes. It thus appears that some antianginal drugs may interfere with the fast Na + channel. To investigate this possibility, we measured the inhibitory effect of 22Na uptake in rat brain synaptosomes and in rat heart cell cultures enriched in muscle cells. Several Ca 2+ antagonists were tested: verapamil, flunarizine, perhexiline, two compounds structurally related to perhexiline: IPS 629: N-(a-methyl-/3-phenylethyl)-2-cyclohexyl-2phenylethylamine (Leclerc et al., 1982) and IPS 672: 3-[4-(2 m e t h o x y p h e n y l ) p i p e r a z i n y l ] - N (phenyl-cyclohexylmethyl)-propionamide (Marciniak et al., 1986, submitted). Two fl-adrenoceptor antagonists, propranolol and practolol, were also tested.

2. Materials and methods

2.1. Preparation of synaptosomes Synaptosomes were prepared from whole rat brain according to the method described by Postma and Catterall (1984). Briefly, the brains of four male Wistar rats were removed and homogenized in cold 0.32 M sucrose, 5 mM K2HPO4, pH 7.4 (10% wet weight/volume) with 10 strokes of a motor-driven teflon-glass homogenizer. The whole procedure was performed at 4°C. The homogenate was centrifuged at 1000 x g for 10 min and the supernatant was retained; the pellet was resuspended in the homogenizing solution (10% volume/volume) and recentrifuged as above. The two supernatants were combined and centrifuged at 17000 x g for 60 min. The resulting pellet was suspended in 9 ml of 0.32 M sucrose, 5 mM K 2 H P O 4, p H 7.4, and was then layered on top of three discontinuous gradients consisting of 7 ml layers of 1.2, 1.0, 0.8, 0.6, 0.4 M sucrose in 5 mM K 2 H P O 4, pH 7.4. The gradients were centrifuged at 100000 x g (SW28 rotor, Beckman centrifuge) for 105 min. The synaptosomes were collected at the 1.0-1.2 M sucrose interface and diluted to 0.32 M sucrose by the dropwise addition of 5 mM K2HPO4, p H 7.4, with constant stirring. The sus-

pension was centrifuged at 40 000 × g for 45 min. The final pellet was resuspended in 5.4 mM KCI, 0.8 mM MgSO 4, 5.5 mM glucose, 50 mM HEPES-Tris (pH 7.4) and 130 mM choline chloride to give a final protein concentration of about 10 mg/ml. The synaptosomes were tested on the same day for 22Na influx. Protein concentration was determined with the method of Lowry et al. (1951).

2.2. Heart muscle cell culture These cells were obtained from the heart of 3 day old rats (Sprague-Dawley) as described previously (Freyss-Beguin et al., 1985). Cells were isolated from the minced hearts by trypsinization at 23 ° C. The enzyme, crystallized twice from pig pancreas (Lab. Choay, Paris) was used at a 0.5-1.0 g/1 concentration in Dulbecco buffered Ca 2+- and MgZ+-free salt solution. Repeated 5 min incubations with trypsin were done until the tissue was almost completely dispersed. Muscle and nonmuscle cells were separated by means of a differential attachment technique, based on the fact that non-muscle cells attached faster to the substrate of the culture flask (Blondel et al., 1971). The trypsinized cells were plated into Leighton tubes equipped with glass shdes at a density of 1.8 X 10 6 cells in 1.8 ml of minimum essential medium (MEM) containing Earle's salts, antibiotic and 10% newborn calf serum. They were incubated at 3 7 ° C for 35-40 min. Fibroblast-like cells, interspersed with 5-10% of muscle cells, thus were attached to the slides. The medium, which contained floating muscle cells interspersed with 10% fibroblast-like cells, was recovered and centrifuged. The cell pellet (muscle cells) was resuspended in MEM and plated at a density of 1.0 X 10 6 cells/2.5 ml MEM. Muscle cells were grown for 8 days at 37 ° C. The gas phase was air without the addition of CO 2. The pH was adjusted every other day with a 4.2% (w/v) N a H C O 3 sterile solution in the presence of phenol red as an indicator. The proliferation of the residual fibroblastlike cells in muscle cell cultures was limited to 10% by two additions, 48 and 72 h after the plating, of 1-/3-D-arabino-furanosyl-cytosine at a final concentration of 10 6 M. The final proportion of

muscle cells in the culture reached 90-92% (Freyss-Beguin and Van Brussel, 1980). The medium was renewed on day 6 and the muscle cells were used on day 8. 2.3. Measurement of 22Na uptake in rat synaptosomes 22Na+ influx was determined according to Tamkun and Catterall (1981) and was evaluated in the presence of 100 /~M protoveratrine B, a veratrum alkaloid which promotes persistent activation of the fast Na + channel gating system. A 50/~l aliquot of the synaptosome preparation was preincubated for 10 min at 37 ° C in a medium containing 5.4 mM KC1, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM HEPES-Tris (pH 7.4), 130 mM choline chloride, bovine serum albumin (BSA) 1 mg/ml, protoveratrine B 100 /~M and the indicated concentration of the drug. After 10 rain, 150 /~l of the 22Na uptake solution were added. This solution, including protoveratrine B 100 #M, contained the same drug concentration as the preincubation medium plus 5.4 mM KC1, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM HEPES-Tris (pH 7.4), 128 mM choline chloride, 2.66 mM NaC1, 5 mM ouabain to block Na ÷ efflux through Na+K+-ATPase and 0.4 ~tCi of carrier-free 22NaC1. After 5 s incubation, 3 ml of ice-cold wash solution (163 mM choline chloride, 0.8 mM MgSO4, 1.8 mM CaCI2, 5 mM HEPES-Tris, pH 7.4, BSA 1 mg/ml) were added to stop the 22Na influx. The mixture was rapidly vacuum-filtered on a 26 mm Whatman G F / C glass fibre disc moistened with wash solution. The filter was washed three times with 3 ml of wash solution. Filter radioactivity was measured in a Packard auto-gamma scintillation spectrometer to determine Na + influx. The inhibition by antianginal and vasodilator drugs of the Na + influx induced by protoveratrine B was tested by determining 22Na+ influx in the presence of increasing concentrations of the drugs. The results were expressed as the percentage of the activity with 100 /~M of protoveratrine B alone. IC50 is the drug concentration which inhibited 50% of the activity of protoveratrine B, 100/~M.

2.4. 22Na uptake in rat heart muscle cells 22Na uptake was determined according to Renaud et al. (1983). The cells were washed three times with incubation medium containing choline chloride 140 mM, KCI 5.4 mM, MgSO4 0.8 mM, CaC12 1.8 raM, glucose 5 raM, HEPES-Tris 25 mM (pH 7.4) then were preincubated for 15 min at 37 o C in this medium. The following were added as indicated, the neurotoxins protoveratrine B (100 /~M) and scorpion toxin (10 #g/ml) from the Leiurus Quinquestriatus which stabilizes an open form of Na + channel (Catterall, 1980), with or without the drugs to be tested. Uptake was started after the incubation medium had been replaced by 1 ml of solution with: choline chloride 130 mM, NaC1 10 mM, KC1 5.4 mM, MgSO4 0.8 mM, CaC12 1.8 raM, glucose 5 mM, HEPES-Tris 25 mM (pH 7.4) enriched with 22NaC1 5 /~Ci/ml, ouabain 0.5 mM and the same concentrations of neurotoxins or drugs as in the preincubation medium. After 1 min incubation, the cells .were washed four times with 3 ml of cold wash m6dium (choline chloride 145 raM, MgSO4 0.8 raM, CaC1 z 1.8 raM, Tris 25 mM, pH 7.4). The cells were digested in 0.1 N NaOH and the radioactivity was measured in a Packard auto-gamma scintillation spectrometer to determine 22Na uptake. Protein concentration was determined with the method of Lowry et al. (1951). The rate of 22Na uptake induced by protoveratrine B and scorpion toxin was obtained by subtracting the 22Na uptake rate in the absence of neurotoxins from that measured in the presence of 100/~M protoveratrine B and 10/~g/ml scorpion toxin. The inhibition by antianginal drugs of the 22Na uptake induced by protoveratrine B and scorpion toxin was tested by determining the 22Na uptake in the presence of increasing concentrations of the antianginal drugs. The antianginal drugs had no effect on 22Na uptake measured in the absence of protoveratrine B and scorpion toxin. The results were expressed as the percentage of the maximal activity obtained with protoveratrine B (100/xM) and scorpion toxin (10 /~g/ml). ICs0 is the drug concentration which inhibited 50% of the activity of protoveratrine B, 100 /~M and scorpion toxin (10/~g/ml).

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2.5. Drugs 4)

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-~ )~100 ~o The drugs used were obtained from the following sources: verapamil HC1 (Biosedra), flunarizine HC1 (Janssen), perhexiline maleate (Merrell), propranolol HC1 and practolol (ICI Pharma), protoveratrine B (Sigma), scorpion toxin from Leiurus Quinquestriatus (Sigma), citrate-free tetrodotoxin (Calbiochem). The IPS derivatives were synthesized in our laboratory by G. Marciniak, IPS 629: N-(amethyl-/3-phenylethyl)-2-cyclohexyl-2, IPS 672: 3[4-(2-methoxyphenyl)piperazinyl]-N-(phenylcyclohexylmethyl) propionamide. 22NaCI was purchased from New England Nuclear Corporation. All other standard laboratory reagents were of analytical grade. Protoveratrine B was dissolved in hydrochloric acid (3 mM in HC1 2%), perhexiline (1 mM) was dissolved in 100% ethanol, flunarizine (1 mM) was dissolved in 0.1 M tartaric acid. Scorpion toxin was prepared according to Catterall (1976): 1 mg/ml in distilled water. Once dissolved, all drugs were diluted to the desired concentration with the buffer used in the ZZNa uptake study. These solvents, at the concentrations used, did not affect 22Na uptake.

3. Results

3.1. Characterization of 22Na uptake in rat heart muscle cells Figure 1 (main figure) shows an example of the time course of 22Na uptake in cultured heart muscle cells under four sets of conditions. Without drugs, 22Na uptake increased slowly to reach a maximum of 20 nmol/mg protein at 2 min of incubation. In the presence of 100 /~M protoveratrine B, or scorpion toxin (10 /~g/ml) alone, the 22Na uptake was not significantly different from that without drugs. The addition of 10/~g/ml scorpion toxin plus 100 /~M protoveratrine B stimulated the 22Na uptake rate which rose sharply to a maximum of 115 nmol/mg protein after 4 min of incubation. Figure 1 (inset) shows the dose-response curve for the effect of tetrodotoxin on the 22Na uptake induced by protoveratrine B

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Fig. 1. The effectsof neurotoxinson 22Na uptake rates in rat heart musclecells in culture. Main figure: timecourseof 2ZNa uptake in the presenceof ouabain(0.5 mM),• withouttoxins, + with protoveratrineB (100 ~tM),[] with scorpion toxin (10 ~g/ml), zxwith protoyeratrineB (100 ~tM)plus scorpiontoxin (10 /Lg/ml). Inset: inhibition by tetrodotoxin of the 22Na uptake inducedby protoveratrineB plus scorpion toxin.

100 /~M and scorpion toxin 10 /~g/ml. Tetrodotoxin inhibited the uptake in a dose-dependent manner at concentrations between 1 and 100/~M. The half-maximum effect of tetrodotoxin was observed at 11 /LM as reported by Renaud et al. (1983). The basal uptake values and stimulated 22Na uptake values were higher than those obtained by Renaud et al. (1983; 1986) who used rat heart cell cultures containing unseparated muscle and non-muscle cells. The present results may have been due partly to good enrichment with muscle cells by means of the differential attachment technique described above and partly to the use of an antimitotic drug to impede fibroblast overgrowth.

3.2. Inhibition of 22Na uptake on rat synaptosomes Figure 2 shows the dose-response curves of rat brain synaptosomes for the inhibition by the antianginal drugs verapamil, flunarizine, perhexiline, IPS 629, IPS 672, propranolol and practolol of the 22Na uptake induced by protoveratrine B (100 /~M). All drugs inhibited this uptake in a dose-dependent manner at concentrations ranging from 1

/~M to 10 mM. All the drugs gave 100% inhibition. IPS 672 was the most potent and practolol the least potent drug. Flunafizine, IPS 629, perhexiline and propranolol had similar effects at doses between 3 and 100/tM. Verapamil was less active between 10 /~M and 1 mM. The dose-response curve for practolol first showed an increase in 22Na uptake at 100 and 300 /~M then a rapid inhibition at doses between 300 #M and 10 mM. 3.3. Inhibition of 22Na uptake in rat heart muscle cells in culture

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Figure 3 shows the dose-response curves for the effect of the antianginal drugs on the 22Na uptake induced by protoveratrine B (100/~M) and scorpion toxin (10 #g/ml) in rat heart muscle cells in culture. As in synaptosomes, all drugs inhibited this uptake in a dose-dependent manner and all except practolol produced 100% inhibition. Because of its poor solubility at high concentrations, practolol could not be tested at doses higher than 10 mM. The most potent drug was IPS 672 and, as with synaptosomes, the least potent was practolol. Inhibition was obtained at concentrations between 1 and 100 /~M, except for practolol which was effective at high doses (1-10 mM).

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Fig. 3. Inhibition by antianginal drugs of the 22Na uptake induced by protoveratrine B and scorpion toxin in rat heart muscle cells in culture. 22Na uptake was measured in the presence of 100/~M protoveratrine B plus 10/~g/ml scorpion toxin; the concentrations of zx verapamil, • flunarizine, + perhexiline, • IPS 629, • IPS 672, [] propranolol and O practolol are given on the abscissa. Each point is the mean of 2 determinations. S.E.M. was never greater than + 15%.

The IC50 for the inhibition of n N a uptake in cardiac muscle cells and synaptosomes are shown in table 1. IPS 672 was the most active compound on synaptosomes (IC50 = 2.0 × 10 -6 M) being nearly 10 times more potent than IPS 629, flunarizine and perhexiline (IC50 = 1.4 × 10 -5 and 3.0 x 10 -5 M) and 55 times more potent than verapamil (IC50 = 1.1 × 10 -4 M). Propranolol was

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TABLE 1

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Inhibition of the nNa uptake induced by protoveratrine B in rat brain synaptosomes and by protoveratrine B and scorpion toxin in rat heart muscle cells in culture. ICso is the molar concentration of antagonist which inhibited the maximum effect of protoveratrine B (100 /~M) or protoveratrine B (100 /~M)+scorpion toxin ( 1 0 / t g / m l ) by 50%. Means+ S.E.M. for 2 or 3 determinations.

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Fig. 2. Inhibition by antianginal drugs of the 22Na uptake induced by protoveratrine B in rat synaptosomes. 22Na uptake was measured in the presence of 100 laM protoveratrine B; the concentrations of t, verapamil, • flunarizine, + perhexiline, • IPS 629, • IPS 672, [] propranolol and O practolol are shown on the abscissa. Each point is the mean of 3 determinations. S.E.M. was never greater than + 10%.

Drugs

IC50 muscle cells mol/1 (n = 2)

IC5o synaptosomes mol/1 (n = 3)

Verapamil Flunarizine Perhexiline IPS 629 IPS 672 Propranolol Practolol

(2.2+0.9) (1.3+3.4) (5.4 + 2.6) (3.0+1.8) (2.4+0.7) (9.7 + 6.3) (6.0+0.1)

(1.1+1.3) (1.4+0.4) (3.0 + 0.2) (1.4+0.5) (2.0+0.7) (3.6 + 1.0) (3.0+1.7)

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Fig. 4. Relationship between the inhibition by antianginal drugs of 22Na uptake in rat brain synaptosomes and in rat heart muscle cells in culture (r = 0.9293, P < 0.01). IC50 is the molar concentration of antagonist which inhibits the maximum effect of protoveratrine B (100 #M) in synaptosomes or protoveratrine B (100 #M)+ scorpion toxin (10 ~g/ml) in rat heart muscle cells by 50%.

100 times more potent than practolol which was the weakest inhibitor with a ICs0 = 3.0 × 10 -3 M. IPS 672 and IPS 629 were the most active drugs on heart muscle cells (ICs0 = 2.4 × 10 - 6 and 3.0 × 10 - 6 M respectively). They were 2-fold more active than perhexiline and more than 5 times more potent than flunarizine and verapamil (ICs0 = 1.3 x 10 -5 and 2.2 × 10 -5 M respectively). As on synaptosomes, propranolol was 100 times more active than practolol which was also the less active inhibitor (ICs0 = 6.0 x 10-3 M). Perhexiline, IPS 629, verapamil and propranolol were about 5 times more active on rat heart muscle cells than on synaptosomes. In contrast, IPS 672 and flunarizine had the same effects on both tissues, while practolol was 2-fold more active on synaptosomes. The ICs0 for cardiac muscle cells and for brain synaptosomes correlated well (r = 0.9293, P < 0.01) (fig. 4).

4. Discussion

The results presented here provide evidence for the hypothesis that antianginal drugs such as

verapamil, flunarizine, perhexiline, IPS 629, IPS 672, propranolol and practolol inhibit :ZNa uptake in rat brain synaptosomes and in rat heart muscle cells in culture. Our results showed a good correlation between the inhibitory effects of antianginal drugs on rat brain synaptosomes and on heart muscle cells in culture. There were some differences between the two tissues: rat heart cells have been described as resistant to tetrodotoxin (Renaud et al., 1983) when c o m p a r e d with synaptosomes; protoveratrine or veratridine (Renaud et al., 1983) themselves have no effect on 22Na uptake in rat heart cells in contrast to synaptosomes. In spite of these discrepancies, our results suggest that the potencies of antianginal drugs on the fast Na + channel can be evaluated equally well on a synaptosomal preparation and on cultured heart cells. We have now shown that some antianginal drugs inhibit the fast Na + channel. It can be noted that verapamil, a class II calcium antagonist (Spedding, 1985) although it exhibited a better Ca z+ blocking effect, was less active on 22Na uptake than the arylalkylamines. Our results for verapamil contrasted with the observations of Fosset et al. (1977) who found no inhibition by verapamil or by its methoxy derivative, D 600, at concentrations up to 100 #M, on veratridinestimulated 22Na uptake in chick embryo heart cells in culture. In contrast to Fosset (1977), and like us, Galper and Catterall (1979) reported that verapamil and D 600 inhibited veratridine-induced sodium uptake in chick embryo heart cells in culture. Frelin et al. (1982) obtained similar results on rat synaptosomes with verapamil and D 600, which blocked the fast N a + channel with respective ICs0 of 15 and 60 /tM. Electrophysiological experiments (Bayer et al., 1975) showed that (+)-verapamil (10 #M) had an inhibitory effect on the fast sodium current in ventricular myocardium and confirmed the interaction of verapamil with the fast N a + channel. Flunarizine, which belongs to the class III calcium antagonists (Spedding, 1985) was less active on the Ca 2+ channel than verapamil (Fleckenstein, 1985) but it inhibited 22Na uptake more than verapamil, particularly in brain synaptosomes. Flunarizine has also been described as a

potent inhibitor of [14C]guanidine uptake, which provides a good probe for measuring the sodium flux through the fast Na + channel (Pauwels et al., 1986). Flunarizine was described as one of the best protectors of veratrine-induced calcium overload in cardiac isolated myocytes (Verdonck et al., 1986). This effect could be linked with the inhibition of Na + influx which reduced Ca 2+ overload through the Ca2+-Na + exchange. The perhexiline derivatives, IPS 629 and IPS 672, were the most active inhibitors of ZZNa uptake in rat heart muscle cells and in rat synaptosomes. The concentrations of perhexiline, IPS 629 and IPS 672 which had a calcium antagonist effect (Leclerc et al., 1982, data not published for IPS 672) were in the same range (1-10 /~M) as the concentrations which inhibited the fast Na + channel in the heart muscle cells. Horowitz et al. (1982) described the inhibition by perhexiline of 45Ca influx in chick embryo ventricular cells at the same doses that inhibited Na influx. Our results on the inhibition of sodium uptake by calcium antagonists compared with their abilities to antagonize the calcium channel suggest that potent inhibitors of sodium uptake are poor calcium antagonists and, conversely, weak inhibitors of sodium uptake are more potent as calcium antagonists. The cross-reactivity between Ca 2+ channel and fast Na + channel inhibitors was also established by Spedding and Berg (1985) who showed that local anesthetics inhibited the Ca 2+ channel in smooth muscle. The fl-adrenoceptor antagonist, propranolol, also inhibited 22Na influx in brain synaptosomes and rat heart muscle cells. This is consistent with the data of Matthews and Baker (1982) which showed that propranolol inhibits 22Na uptake in rat brain membranes (ICso = 6.5 ttM) and of Tarr et al. (1973) who demonstrated that propranolol 30 /~M suppresses the inward sodium current by blocking the sodium channel in dog atrial muscle. Practolol is 100 times less active than propranolol in inhibiting 22Na uptake both in synaptosomes and in heart muscle cells. A similar difference has been observed in the fl-adrenoceptor antagonist properties of these compounds (Bieth et al., 1980) and in a variety of their effects

e.g. antiarrhythmic action, local anaesthetic action or membrane-stabilizing effect (Hellenbrecht et al., 1973). All the antianginal drugs tested here inhibit the binding of [3H]batrachotoxinin A20a benzoate (McNeal et al., 1985; Grima et al., 1986; Pauwels et al., 1986). The arylalkylamines were the most potent of all the drugs tested, with IC50 values in the range of 27-600 nM. Verapamil was 10 times less potent than flunarizine. Propranolol inhibited batrachotoxin binding at a concentration of 10 ~M. Practolol was considered as inactive. Our results agree fully with those of McNeal et al. (1985), confirming that antianginal drugs act on the fast Na ÷ channel. We were not able however to correlate the inhibition of Na uptake by synaptosomes or by heart muscle cells in culture with the inhibition of batrachotoxin binding in rat synaptosomes or rat heart membranes. The two experimental models are in fact different since [3H]batrachotoxinin is a specific ligand used to characterize receptor sites on the fast Na + channel while 22Na uptake indicates the total activity of the fast Na + channel gating system. The concentrations of antianginal drugs that inhibited 22Na uptake or the fast Na + current are higher than the concentrations that inhibited the binding of [3H]batrachotoxinin A20a benzoate. We have no explanation for this discrepancy. The measurement of 22Na uptake also required high concentrations of agonist, i.e. protoveratrine stimulated 22Na uptake between 1 and 100 /~M while the Na-related inotropic effect on atria is observed at 0.01-1 ~M (Decker et al., 1986). Pauwels et al. (1986) have recently shown that the [14C]guanidine uptake in rat brain was also a good model for studying the fast Na ÷ channel. Only slight differences were noted between the doses of drugs that inhibited the binding of [3H]batrachotoxinin and [14C]guanidine uptake. This technique could provide a more sensitive model than 22Na uptake. It is still not clear how these antianginal drugs, which belong to different chemical and pharmacological groups, interfere with the fast Na + channel. But, in short, the results presented in this work may provide useful new information on the common effect of various antianginal drugs on the fast Na ÷ channel in rat brain and heart.

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