Actions of D 600 on isolated rabbit aorta

Gen. Pharraac. Vol. 13, pp. 343 to 346, 1982 Printed in Great Britain. All rights reserved

0306-3623/82/040343-04503.00/0 Copyright ~ 1982 Pergamon Press Ltd

ACTIONS OF D 600 O N ISOLATED RABBIT AORTA M. AUGUET a n d F. V. DEFEUDIS* Institut Henri Beaufour, 72 Avenue des Tropiques, 91940 Les Ulis, France (Receired 30 Not'ember 1981)

Abstraet--l. The actions of D 600 (10-9-10 -`* M) on various phases of NE- or CaZ+-induced contractions of rabbit isolated aorta were determined in Ca 2 +-free medium. 2. D 600 inhibited the slow phase of contraction with ECs0 ~ 3 x 10 -s M, but affected the rapid phase of contraction only at 10 -4 M. 3. D 600 increased the relaxation that followed the rapid phase of NE-induced contraction in Ca 2 + free medium with ECs0 ~ 1.5 x 10-5 M. 4. The method described might be useful for further studies on Ca 2 +-antagonists.

INTRODUCTION The systematic study of organic calcium-antagonists ("calcium influx blockers" or "slow-channel blockers"), such as D 600 (methoxy-verapamil), using preparations of isolated vascular smooth muscle (e.g. rabbit aortic strips) generally leads the experimenter to d e m o n s t r a t e inhibitory effects of these substances on contractions induced by KCI or CaCI2 (e.g. Massingham, 1973; Schtimann et al., 1975; K a z d a et al., 1980). However, calcium-antagonists appear to be relatively less effective on contractions induced by norepinephrine (NE) (e.g. Massingham, 1973; Schiimann et al., 1975). The contractile response induced by N E has been separated into two phases, a fast (phasic) phase which is believed to be dependent on intracellular calcium, and a slow (tonic) phase which is believed to be dependent on extracellular calcium (Bohr, 1963, 1973; van Breemen, 1969; Sitrin & Behr, 1971 ; G o d f r a i n d & Kaba, 1972; Bolton, 1979). Using a technique derived from the l a n t h a n u m method (van Breemen et al., 1973), we have shown that D 600 does produce an inhibition of b o t h phases of the NE-induced contraction of rabbit isolated aorta. The results are presented herein. MATERIALS AND METHODS White male New Zealand rabbits (EVIC-CEBA), 1.82.5 kg, were killed by cervical dislocation. The thoracic aorta was rapidly excised, placed in medium (see below) at 37~C, and cut into spiral strips (3--4mm wide, 9--14ram long) (Furchgott & Bhadrakom, 1953). Each strip was suspended vertically in a 20-ml organ bath, its lower end being attached with a linen thread to a glass bubbler fixed to the bottom of the bath. and its upper end being connected to a force-displacement transducer (Narco BiD-Systems, F 60). Isometric contractions were recorded using a physiograph INarco BiD-Systems, MK IV). The medium that was used had the following composition (raM): NaCI, 154.0; KCI, 5.4; CaCI z, 2.4; NaHCO3, 6.0; glucose I1.0. The calcium-free medium had the same composition, " Present address: U.P.S.A,, 128 rue Danton, B.P. No. 325, 92506 Rueil-Malmaison Cedex, France. 343

except that the CaCI2 was replaced by 0.1 mM of disodiumethylenediaminetetraacetate (EDTA; Sigma Chem. Co.). Both media were continuously gassed with 95% O2/5% CO2, and their pH was about 7. Aortic strips were subjected to an initial resting tension of 2 g, and were equilibrated in the medium for 1 hr during which the medium was renewed and the tension was readjusted approximately every 10 rain. Then, NE (1-noradrenaline-l-hydrogentartrate; Fluka) was added to the medium to provide a concentration of 10- 5 M. 8 min after the addition of NE, when the muscular contraction was at a maximum, 0.3 mM of CaCI 2 was added and 4 min later the preparation was washed with calcium-free medium. After three washes at l-min intervals, a 40-min rest period was allowed, during which the preparation was washed every 10min with calcium-free medium and the tension was readjusted. 8 rain later, NE was again tested, and after another 8 min interval 0.3 mM CaCI2 was added. A final reading was taken between 13 and 15 rain after the application of CaCI 2. D 600-HCI (Gallopamil-hydrochloride; Knoll AG) was added to the calcium-free medium 5 min before the second addition of NE. This procedure is illustrated in Fig. 1. RESULTS Results presented in Fig. 1 show that under control conditions (absence of D 600) the protocol used reveals clearly the two phases of the contractile response of the rabbit aortic strip to NE, a n d that addition of 0.3 m M CaCI2 restores the slow phase of the contraction (see Hester & Carrier, 1978). Calculated ratios provided in Table 1 also show that this protocol gives consistent results --the ratios between points B, C or D and point A of the contraction in the absence of D 600 are reproducible (see also Fig. 1). Addition of D 600 did not influence the ratio B/A except at the 1 0 - 4 M concentration, but all other ratios were affected by D 600 in a concentrationdependent fashion (Fig. l ; Table l). In order to quantify the effect of D 600, the ratios obtained with control aortas (considered to be fixed parameters) were c o m p a r e d with ratios for aortas treated with the drug (see Fig. 2). For the ratios D/A or D/B the general shape of the curves indicates a superposition of two effects, one sigmoidal, the other

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Fig. I. Representative recordings of isometric contractions of rabbit aortic strips by norepinephrine (NE, 10- 5 M) in control medium and in Ca 2 *-free, EDTA medium in the absence or presence of D 600 (10-9-10 -* M), and the effect of Ca 2 + addition after blocking the slow phase of NE-induced contraction with D 600. (A) 8 min after addition of NE; no effect of CaCI2 in control medium; (B) peak of fast phase of contraction after addition of NE in Ca 2*-free medium; (C) 8 rain after addition of NE in Ca 2 +-free medium; addition of CaCI2; (D) 13-15 min after addition of CaCI2, a time corresponding to the maximal response to Ca 2 +. Ratios calculated using A, B, C and D are provided in Table I and are graphically displayed in Fig. 2.

Table 1. Ratios* for contraction of rabbit aortic strips in the presence and absence of D 600 Concentration of D 600 (M) 0 10- 9 10 -a 10 - ~' 10 -6 10 -s 10 -4

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D/B

0.61 +0.04 0.56 0.69 0.77 0.54 0.55 0.66 0.53 0.57 0.55 0.45 0.66 0.45 0.46

0.36 +0.04 0.33 0.39 0.50 0.33 0.23 0.34 0.21 0.27 0.20 0.22 0.20 0.13 0.13

1.27 +0.06 1.12 1.51 1.12 i.05 0.49 0.53 0.28 0.36 0.16 0.25 0.20 0.08 0.09

2.08 +0.06 2.00 2.20 1.45 1.94 0.90 0.80 0.53 0.64 0.29 0.57 0.31 0.18 0.19

(D-C)/A 0.91 -+0.05 0.79 1.13 0.62 0.71 0.26 0.19 0.07 0.09 -0.04 0.04 0.00 -0.05 -0.04

* S¢¢ Fig. I for an explanation of A, B, C and D. Means + SEM for 5 preparations tested in the absence of D 600: individual values for preparations tested with D 600.

linear (Fig. 2a, b). Ratios used for Fig. 2c (D-C/A) and for Fig. 2d (C/A) permit a separation of these two phenomena. F r o m the plot shown in Fig. 2c an ECho of 3 x l0 - s M can be graphically determined for the action of D 600 on the slow phase of NE-induced contraction. F r o m the results shown in Fig. 2d, which correspond to the effect of D 600 on the relaxation which follows the rapid phase of the contraction, an ECso of 1.5 x 10-s M can be determined.

DISCUSSION

Even though D 600 does not appear to interact specifically with NE-receptors (e.g. Schiimann et al., 1975), the method described here permits the systematic study of three aspects of the action of D 600 o n smooth muscle contraction. (!) Its effect on the rapid phase of the contraction, which is dependent upon intracellular calcium (denoted by B in Fig. I), can be determined. In accord with the results of other workers (e.g. Karaki et al., 1979), D 600 had very little effect on this phase, except at a very high concen-

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(d) Fig. 2. Calculated ratios showing the effects of D 600 on various phases of contraction of isolated aortic strips. The values used to calculate these ratios are shown in Table 1; see Fig. 1 for explanation of A, B, C and D. Graphs of the ratios D/A or D/B vs log[D 600] indicate that D 600 exerts two different effects on contraction; a graph of the ratio D-C/A vs log[D 600] indicates that D 600 inhibits the slow phase of contraction with ECho ~ 3 x 10 -8 M; a graph of the ratio C/A vs log[D 600] indicates that D 600 increases the extent of relaxation which follows the rapid phase of contraction in Ca2+-free medium with ECs0 -~ 1.5 x 10-5 M. Each plotted point represents the ratio of the value for an individual aortic strip in the presence of D 600 and the mean of 5 values from 5 aortic strips in the absence of D 600.

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Acknowledgement- We are grateful to Caroline Charras for translating the first draft of this manuscript from French to English.

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tration (10 -4 M). (2) In contrast, the relaxation which followed this rapid phase (denoted by C in Fig. 1) was more sensitive to D 600, but this effect of D 600 was clear only at concentrations higher than 10-6 M. This effect of D 600 could be due to a local anaesthetic-like action (Golenhofen & Hermstein, 1975; Rahwan et al., 1979), or to an increase in the internal re-binding of Ca 2÷ to those storage sites which are involved in the relaxation (Deth & van Breemen, 1974). (3) Most importantly, the action of D 600 on the slow phase of contraction can be shown with the method described, the ECso estimated for D 600 (3 × 10- 8 M) being in agreement with results obtained for its inhibition of C a ' +-induced contraction of aortic smooth muscle in potassium-rich medium (Schiimann et al., 1975). This method might be useful for further characterizing the actions of calcium-antagonists.

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REFERENCES -4

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346

M. At:Gtn-t and F. V. I)I!FIct;DIs

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