Ca2+ channel blocking activity of lacidipine and amlodipine in A7r5 vascular smooth muscle cells

European Journal of Pharmacology - Molecular Pharmacology Section, 244 (1993) 139-144

139

/c3 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/$06.00

EJPMOL 90394

C a 2+

channel blocking activity of lacidipine and amlodipine in A7r5 vascular smooth muscle cells Santi S p a m p i n a t o a, T i z i a n a B a c h e t t i a, L u c i a C a r b o n i ~', E m i l i a n g e l o R a t t i b, F r a n k T h . M . V a n A m s t e r d a m b and Sergio F e r r i ~'

"Department of Pharmacology, University of Bologna, Bologna, Italy, and t, Glaxo Research Laboratories, Verona, Italy

Received 14 September 1992,accepted 6 October 1992

Inhibition of the K+-stimulated increase in cytosolic free Ca 2+ by a series of 1,4-dihydropyridines was evaluated in A7r 5 vascular smooth muscle cells loaded with the fluorescent Ca 2+ indicator fura-2 acetoxymethyl ester. The ICs0 of the drugs, added to suspended cells 3 min before 150 mM KCI, gave the following order of potency: lacidipine (2.76 nM) > nitrendipine (3.81 nM) > amlodipine (4.56 nM) > nifedipine (10.08 nM). A7r 5 cells were also exposed to the 1,4-dihydropyridines, at their ICs0, for 25 min, and then repeated washout cycles were performed before adding KCI. The C a 2+ channel blocking activity of nifedipine and nitrendipine gradually diminished, disappearing after four washout cycles 25, 55, 115 and 175 min after drug treatment. Amlodipine and lacidipine displayed slow onset and offset of antagonism, their activity becoming stronger with time, in spite of the repeated washes. [3H]Lacidipine was avidly and promptly entrapped in A7r 5 cells and was not removed by washout. However, its potency as a Ca 2+ channel blocker was not directly related to the amount of drug locked in the cell since it increased with time, indicating that lacidipine binds to the lipid bilayer of the cell membrane and then gradually diffuses towards a specific binding site. This model can, therefore, predict the Ca 2+ blocking properties of 1,4-dihydropyridines with slow onset and offset of antagonism and could be employed to evaluate compounds selective for vascular smooth muscle. Smooth muscle (vascular); Ca 2+ influx; A 7 r 5 cells; Lacidipine; Amlodipine; 1,4-Dihydropyridines

I. Introduction

Voltage-dependent Ca 2+ channels play an important role in excitation-contraction coupling in cardiac and smooth muscle (Meldolesi and Pozzan, 1987). The 1,4-dihydropyridines potently inhibit or activate high threshold, long lasting Ca 2+ channels (the L-type of voltage dependent channel) expressed in these tissues (Glossmann et al., 1987; Hofmann et al., 1987). These agents include Ca 2+ antagonists valuable for the treatment of several cardiovascular diseases (StruykerBoudier et al., 1990). L-type channels are found mainly in the skeletal muscle, in the heart and in smooth muscle. The dihydropyridine-sensitive Ca 2÷ channel, recently isolated from skeletal muscle (Tanabe et al., 1987), is currently believed to consist of a central ion channel-forming element interacting with three other non-covalently associated subunits (Catterall et al.,

Correspondence to: Dr. Santi Spampinato, Department of Pharmacology, University of Bologna, Irnerio 48, 40126 Bologna, Italy. Fax 39-51-248862.

1989). The a I subunit, which contains the Ca 2+ antagonist binding sites, is the proposed central ion channel-forming component of the complex. The a 1 subunit has recently been cloned from skeletal muscle (Tanabe et al., 1987) and from the rabbit heart (Mikami et al., 1989). Although the two proteins reveal a high degree of homology (66% of amino acids are common to both sequences), there are clear-cut differences, possibly due to the expression of different genes or alternative splicing which may contribute to the tissue specificity of Ca 2+ antagonist action (Slish et al., 1989; Tsien et al., 1991). The 1,4-dihydropyridine derivatives can be classified on the basis of their chemical, receptor binding and pharmacological properties (Vanhoutte and Paoletti, 1987), and display marked differences in their vascular and cardiac sensitivities (Struyker-Boudier et al., 1990). Taking into consideration the tissue selectivity and the duration of action, relevant to the therapeutic purpose, we selected a series of 1,4-dihydropyridines with widely varying kinetic properties and investigated their profiles as Ca 2+ -entry blockers by using the fluorescent chelator fura-2 acetoxymethyl ester (AM) (Grynkiewics et al., 1985; Malgaroli et al., 1987; Tsien, 1988) in A7r 5 cells, an aortic smooth muscle cell line,

140

expressing L-type C a 2+ channels (Sperti and Colucci, 1987). Nifedipine and nitrendipine, two compounds with rapid onset of action, that reach equilibrium with the high-affinity state of the binding site in a short time (Kwon et al., 1990), were compared with amlodipine and lacidipine, both of which have slow kinetics of onset and offset of antagonism in smooth muscle preparations (Burges et al., 1987; Micheli et al., 1990).

2. Materials and methods

2.1. Cell culture The A7r 5 cell line, an embryonic rat aortic smooth muscle line (Kimes and Brandt, 1976), was obtained from the American Type Culture Collection (Bethesda, MD, USA). Ceils were grown in 75 cm 2 plastic culture flasks at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 /zM biotin and 1 /zM lipoic acid in a humidified atmosphere under 5% CO2/95% air. Confluent cell layers were detached by treatment with 0.05% trypsin (Gibco).

2.2. Ca 2 +-sensitive fura-2 fluorescence Cells were harvested by gentle agitation in warm solution B, containing (mM concentrations) 137 NaC1, 5.4 KC1, 0.17 Na2HPO 4, 0.22 KH2PO 4, 5 glucose and 58 sucrose, resuspended (4 x 10 6 cells/ml) in solution C (containing 130 NaCI, 5 KC1, 1.5 CaC12, 1 MgC12, 10 glucose and 20 HEPES, pH 7.4) with additional 0.5 mg/ml bovine serum albumin (BSA) (solution A). The cell suspension was then incubated with 2/xM fura-2 AM for 20 min in a shaking water bath at 37°C. Cells were pelleted by low-speed centrifugation, resuspended in solution C with additional 5 mg/ml BSA and incubated for another 10 min at 37°C. The cells were then centrifuged, resuspended (2 × 10 6 cells/ml) in solution A and kept at room temperature (Berk et al., 1987). Fluorescence was measured on a Perkin-Elmer LS-5 fluorescence spectrophotometer at excitation and emission wavelengths of 340 and 510 nm, 5 and 10 nm slit width, respectively. The cells ( ~ 2 x 1 0 6 / 2 ml/cuvette) were kept in suspension at 37°C with a magnetic stirrer. Depolarization was induced by adding different doses of KCI (50, 75, 100 or 150 mM) to the cell suspension. The change in fluorescence (AF) was calculated from the difference between final (Ff; determined 3 min after addition of KC1) and initial (Fi; determined 30 s prior to KC1) values. Cells were responsive to K+-induced depolarization up to 180 min after loading the fluorescent chelant.

D

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Fig. 1. Experimental protocol followed to evaluate Ca 2+ blocking activity of 1,4-dihydropyridines in fura-2 AM loaded A7r 5 cells after repeated washout cycles. Cells were exposed to the drugs (D) for 25 min and then 1-4 washes (W) were performed before addition of 150 mM KCI (K + ).

Fura-2 leaking out in the meantime was removed by washing the cells with solution A immediately before the fluorescence measurements (data not shown). Fura-2 leaked out of A7r 5 cells at a rate of 3.12 + 0.12%/60 min (n = 8) at 25°C. The effect of the compounds assayed on the K+-induced increase of cytosolic Ca 2+ concentration [Ca2+] i was established by preincubating the fura-2 AM loaded cells with a vehicle or with different concentrations of drugs for 3 min before the challenge with KC1. To assess recovery of the K+-induced increase of [Ca2+]i in treated cells, fura-2 AM loaded A7r 5 cells were exposed to the drugs for 25 min and then washed 1-4 times before adding KCI (150 mM) (see fig. 1). Briefly, cells were centrifuged (460 x g for 5 min) and resuspended in fresh drug-free solution A (2 m l / ~ 2 × 10 6 cells). The dye carried over from each wash was 3.1 + 0.22% (n = 8). Cells were used only once. Loading aortic smooth muscle cells with fura-2 AM and washout cycles did not affect cell viability: 180 min after loading and after 4 washes more than 97% of the cells still excluded trypan blue. IC50 values, defined as the concentration of drug that gives a half-maximal decrease in AF, were calculated using logit analysis. Assuming a uniform distribution of intracellular Ca 2÷, [ C a 2 + ] i w a s calculated as described (Grynkiewics et al., 1985), according to the e q u a t i o n : [Ca2+]i = K D [(F i - Fmin)/(Fmax - Fi)], w h e r e

K D is the apparent dissociation constant of fura-2 for C a 2+ (225 nM), F i is the fluorescence signal in arbitrary units of intact fura-2 AM loaded cells, and Fmin and Fmax are the minimum and maximum fluorescence after addition of 5 mM EGTA + 30 mM Tris and 0.05% Triton X-100 + 6 mM CaC12, respectively. The background autofluorescence, measured in unloaded cells from the same preparation, was subtracted from all the measurements. The complete intracellular hydrolysis of fura-2 AM was verified by recording the emission spectrum of fura-2 from 300 to 600 nm in the loaded cells: the

141

fluorescence peak was only seen at 340 nm at the end of the loading procedure. Moreover, stimulation of the cells did not lead to any loss of intracellularly trapped fura-2 as ascertained by fluorescence measurements (data not shown) 2.3. Chemicals Fura-2 AM (Boehringer Mannheim) was dissolved in dimethyl sulfoxide (DMSO, Aldrich) at a concentration of 1 mM and stored at -20°C. Nifedipine was obtained from Sigma. Nitrendipine, amlodipine (3-ethyl,5-methyl,2-(2-aminoethoxymethyl)4-(2-chlorophenyl)-l,4-dihydro-6-methyl-3,5-pyridinedicarboxylate), lacidipine ((E)-4-(2-(3-(1,1-dimethylethoxy)-3-oxo- 1-propenyl)phenyl- 1,4-dihydro-2,6-dimethyl3,5-pyridinedicarboxylic acid diethyl ester) and [3H]lacidipine (specific activity 21.6 Ci/mmol) were obtained from Glaxo Research Laboratories, Verona, Italy. The 1,4-dihydropyridines were dissolved in absolute ethanol (10 -z M) and then diluted with distilled water to a final maximum concentration of 0.1% (v/v) of ethanol in the cuvette. As control, a vehicle was prepared by diluting absolute ethanol in distilled water (the final concentration of ethanol was 0.1% (v/v) as the solutions of the drugs). Drugs were added to the cuvette by pipetting 10-40 /xl of the stock solution at the appropriate concentration.

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Fig. 3. Concentration dependence of the effect of nifedipine ([]) and nitrendipine ( I ) (panel A) and of lacidipine (©) and amlodipine (e) (panel B) on the increase of [Ca 2+ ]i induced by 150 m M KCI. Drugs were added to cell suspensions 3 min before the depolarizing agent. Data are percentages of the maximal response obtained when a vehicle was added before KCI. Each point is the m e a n + S.E.M. of 3 - 5 determinations. Differences in slopes of the dose-response curves were determined by the test for parallelism as described by Tallarida and Murray (1987) and the following were significant (P < 0.05): lacidipine vs. nifedipine and nitrendipine; amlodipine vs. nifedipine and nitrendipine; nifedipine vs. nitrendipine. Lacidipine vs. amlodipine was not different (P > 0.05).

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All solutions were made daily and protected from light. Experiments were conducted in darkened laboratories.

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2.4. Data analysis

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Pharmacological data were analyzed using a standard set of specific programs (Tallarida and Murray, 1987).

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TABLE 1 o

Inhibition of Ca :+ influx in A 7 r 5 cells by Ca 2÷ channel blockers.

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Fig. 2. Concentration dependence of KCl-evoked increase of [Ca 2 ÷ ]i in ATr 5 cells. KCI was added to cell suspensions and fluorescence changes were followed as described in Materials and methods. The data are expressed as increase in intracellular free Ca 2+ ( m e a n + S.E.M. of six determinations). Basal values for resting [Ca 2÷ ]i were 1 3 6 + 6 n M (mean :l: S.E.M.; n = 4 8 ) . Cells were suspended in solution A (e) or in a Ca2÷-free solution containing 2 m M E G T A (©). For cells suspended in solution A the correlation coefficient and the regression equation of the concentration-response curve were: r = 0.97; y = 0.248x+ 5.32.

Cells were exposed to various concentrations of agents (0.3-75 nM) 3 min before KCI (150 mM). IC50 values were calculated from logit analysis of the data in fig. 1 (Tallarida and Murray, 1987) and are defined as the concentration of drug which inhibited the maximal response by 50% when a ,¢ehicle was added before KCI. Agent

IC50 (nM) +_S.E.M.

Potency relative to lacidipine

Lacidipine Nitrendipine Amlodipine Nifedipine

2.76 + 0.11 3.81 + 0.08 4.56 _+0.12 10.08 + 0.13

1.00 1.06 1.73 2.70

142

3. Results

CELL

Quantitative measurements of [Ca2+] i in fura-2 AM loaded cells indicated a basal level of 136 + 6 nM (mean + S.E., n = 48). Upon stimulation with KC1, there was a rapid and concentration-dependent rise in cytosolic free Ca 2+ (fig. 2). Maximal response was achieved with 150 mM KCI, higher doses causing damage to the cell viability (data not shown). When cells were suspended in a Ca2+-free solution containing 2 mM EGTA, the KCI response was abolished (fig. 2). Nifedipine, nitrendipine, lacidipine and amlodipine added to the A7r 5 cells 3 min before 150 mM KCI produced a dose-dependent inhibition of [Ca2+] i elevation (fig. 3). The IC50 are reported in table 1, the order of potency being as follows: lacidipine > nitrendipine > amlodipine > nifedipine. In a separate set of experiments, A7r 5 cells were incubated for 25 min with the IC50 of each 1,4-dihydropyridine (see table 1) and subsequently washed out 25, 55, 115 and 175 min after drug treatment (fig. 1). The depolarizing agent was added to the cells after the last washout. The activity of nifedipine and nitrendipine was gradually reduced by washing and the dose causing

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Fig. 5. Percentage of [3H]lacidipine (1-10 nM) in A7r 5 cells exposed to the compound for 25 rain. Cells were centrifuged briefly and the radioactivity in the cells or supernatant was counted in a/3-counter. The cells were washed 25, 55, 115 and 175 rain after drug exposure as described in the legend to fig. 1. [3H]Lacidipine was added only once at time 0.

lau ¢,n Z O a.

a 50% reduction in the Ca 2+ influx induced by 150 mM KCI was not effective after 180 min and four cycles of washout (fig. 4). The potency of lacidipine and amlodipine, on the other hand, increased with time (fig.

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75

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The IC50 in A7r 5 cells incubated with different concentrations (0.1-50 nM) of these two compounds for 25 min then washed 4 times was 1.33 nM + 0.11 (n = 4) for lacidipine and 3.07 n M + 0.18 (n = 3) for amlodipine. ATr5 cells were incubated with [3H]lacidipine (1-10 nM) for 25 min and exposed to washes as previously described (see fig. 1). Between 74 and 78% of radioactivity was entrapped in the cells, independent of the time and the dose administered (fig. 5). Less than 5% of [3H]lacidipine was found in the supernatant after pelletting the cells at various times (fig. 5).

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Fig. 4. Effect of nifedipine (o, 10 nM), nitrendipine (e, 3.8 nM), amiodipine ( - , 4.5 nM) and lacidipine ( zx, 2.75 nM) on the increase of [Ca2+]i induced by 150 mM KCI. Drugs were added to the cells left at 25°C for 25 min; these were then briefly centrifuged and resuspended in fresh, drug-free solution A, 25, 55, 115 and 175 min afterwards. The depolarizing agent was added to the cells after the last washout, 30, 60, 120 or 180 min after exposure to the 1,4-dihydropyridines (added only once at time 0) (see fig. 1).

4. Discussion

We evaluated the Ca 2+ channel blocking activity of a series of 1,4-dihydropyridines in A7r 5 vascular smooth muscle cells. These cells are derived from the thoracic aorta of embryonic rats and maintain the characteristic properties of smooth muscle (Kimes and Brandt, 1976).

143

They express dihydropyridine-sensitive, voltage-dependent Ca 2+ channels (Sperti and Colucci, 1987) and have proved useful as model systems for studying the regulation of [Ca2+] i levels by phorbol esters through L-type channels (Fish et al., 1988; Vigne et al., 1988). Quar et al. (1988) and Schmid et al. (1989) employed these clonal cells to assess new Ca 2÷ channel blockers by binding studies and 45Ca2+ flux experiments. We employed the Ca2+-sensitive fluochrome, fura-2 AM, which is widely used to monitor [Ca2+]i in a variety of tissues and preparations (Malgaroli et al., 1987) including arterial smooth muscle cells (Goldman et al., 1990). Adopting this sensitive technique, we calculated the IC50 of nifedipine, nitrendipine, lacidipine and amlodipine added to the cells 3 min before the depolarizing agent, KCI. The order of potency was as follows: lacidipine > nitrendipine > amlodipine > nifedipine and the values were of the same order of magnitude as in isolated organs (Burges et al., 1987; Micheli et al., 1990). Amlodipine and lacidipine require several hours to achieve complete equilibration leading to an underestimation of their activities in these conditions. Therefore, having checked that viability and responsiveness to KC1 are maintained in cells incubated with fura-2 AM up to 180 min, we evaluated the Ca 2+ blocking activity of the 1,4-dihydropyridines added to the ATr5 cells for 25 min and then repeatedly washed before adding KC1. In this case the potency of lacidipine and amiodipine increased with time in spite of the washout, the former being the more potent. The activity of nifedipine and nitrendipine gradually diminished with time, in agreement with the rapid rate of onset and offset of blocking activity (Kwon et al., 1990). Adopting this experimental model it was not possible to incubate the fura-2 AM loaded A7r 5 cells with the 1,4-dihydropyridines for different periods of time (30180 min) and then add the depolarizing agent directly. In fact, the cells incubated for more than 30 min have to be washed prior to estimation of fluorescence signals to remove the fura-2 which has leaked out in the meantime, since this could affect the fluorescence determinations. The present findings are in line with previous reports that on isolated organs, both lacidipine and amlodipine have a remarkably slow onset of action and their effects are reversed only very slowly after drug washout (Burges et al., 1987; Micheli et al., 1990). Therefore, our model could serve to predict the Ca 2+ channel blocking properties of 1,4-dihydropyridines with slow onset and offset kinetics and could be used as an alternative to in vitro preparations for compounds selective for the vascular smooth muscle. The particular properties of amlodipine have been ascribed to the basic aminoethoxymethyl side chain in the C-2 substituent but not to its lipophilicity (Burges et al., 1987). This was also ascertained by Galletti et al.

(1991), who evaluated analogs of tiamdipine, a compound related to amlodipine (Kwon et al., 1990). Lacidipine is an ortho-substituted 4-aryl-l,4-dihydropyridine, different from amlodipine and from neutral 1,4-dihydropyridines such as nifedipine; Herbette et al. (1991) recently reported that it has a very high membrane partition coefficient into the lipid phase. The ter-butyl side chain could act as an anchor to lock it in the cell membrane's lipid bilayer. These findings are in agreement with our observation that [3H]lacidipine is avidly and promptly entrapped in A7r 5 cells. However, the potency of this drug is not directly related to the amount locked in the cell since it increased with the exposure time, in spite of washes. Therefore, our results offer support to the suggestion by Rhodes et al. (1985), that 1,4-dihydropyridines bind to the lipid bilayer of the cell membrane and then diffuse towards a specific receptor site.

Acknowledgements This study was supported in part by a research grant and a fellowship to T.B. from Glaxo S.p.A. (Verona, Italy) and by C.N.R. Target Project 'Biotechnology and Bioinstrumentation'.

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