European Journal o f Pharmacology, 57 (1979) 57--67 © Elsevier/North-Holland Biomedical Press
57
ACTION OF INDAPAMIDE ON EXCITATION--CONTRACTION COUPLING IN VASCULAR SMOOTH MUSCLE JEAN MIRONNEAU * and YVES-MICHEL GARGOUIL
Laboratoire de Physiologie Cellulaire, Universit~ de Bordeaux 2, et Laboratoire de Physiologie Animale, Universitg de Poitiers, France Received 24 January 1979, revised MS received 27 March 1979, accepted 4 April 1979
J. MIRONNEAU and Y.M. GARGOUIL, Action o f indapamide on the excitation--contraction coupling in vascular smooth muscle, European J. Pharmacol. 57 (1979)57--67. The effects of indapamide on electrophysiological and mechanical parameters of longitudinal smooth muscle strips isolated from mammalian portal vein were studied by means of a double sucrose gap method associated with a photoelectric device for recording contraction. Indapamide (10 -4 M) reduced both the amplitude of the action potential and the contraction. The calcium inward current decreased and consequently the phasic contraction was also reduced. The potassium outward current was diminished while the tonic contraction was not modified significantly. The depressant nature of the indapamide response could counterbalance the stimulating action of angiotensin II but not that of noradrenaline. The results suggest that indapamide acts primarily on the plasma membrane of vascular smooth muscle by reducing the transmembrane calcium current, although a secondary decrease in the intracellular bound calcium could not be completely excluded. Vascular smooth muscle
Ionic currents
Contraction
1. Introduction Indapamide (chloro-4-N-(methyl-2-indolinyl-1)-sulphamoyl-3-benzamide) is a new drug which possesses antihypertensive and diuretic properties (Beregi, 1977). It has been shown that indapamide decreased peripheral arterial resistance as evaluated by analysing the carotidogram (Canicave and Lesbre, 1977) and measuring blood pressure (Finch et al., 1977); these results suggested a possible effect of indapamide on the contractility of vascular smooth muscle. By using a double sucrose gap device, the electrical properties of smooth muscle cells can be investigated with external electrodes. This method has been applied to several visceral smooth muscles (Anderson, 1969; * Present adress: Dr. J. Mironneau, Laboratoires de Physiologic Cellulaire et de Cytologic, Avenue des Facult~s, 33405 Talence Cedex, France.
Indapamide
Mironneau, 1973; Kao and McCullough, 1975; Vassort, 1975) and, more recently, to vascular smooth muscle (Daemers-Lambert, 1976; Mitonneau and Savineau, 1977). Our experiments were designed to study the relations between contraction, ionic currents and transmembrane potentials in mammalian portal vein. The effects of indapamide on excitation-contraction coupling in portal vein were studied by assuming that the drug may act at the cellular level. A preliminary report of part of this work has already been published (Gargouil and Mironneau, 1977).
2. Materials and methods
2.1. Preparation Isolated longitudinal strips (50--150 #m in diameter, 3--4 mm in length) from the portal vein of rabbits were used in these
58 experiments. After a short healing period (about 15--30 min) in the reference solu. tion, the preparation was ready for current or voltage clamping. 2.2. Solutions
The physiological solutions had the following composition: (a) Reference solution (mM): NaC1 130; KC1 5.6; CaC12 2; MgCI2 0.24; glucose 11. The solution was aerated with 02 and buffered with Tris-HC1 (8.3 mM) at pH 8. (b) The high potassium solution was prepared b y substituting NaC1 for KC1 in equimolar amounts (135,6 raM). (c) The following inhibitors of permeability were used: manganese chloride (5 mM) and D600 (a-isopropyl-a- [ (N-methyl-N-homoveratryl)-~-aminopropyl] -3,4,5-trimethoxyphenylacetonitriteHCI, 0.5 mM) considered to be inhibitors of the calcium inward current (Abe, 1971; Mironneau, 1973; Fleckenstein, 1977) while t e t r a e t h y l a m m o n i u m chloride (20 mM) is k n o w n to inhibit the potassium current (Armstrong, 1966). In order to obtain a calcium-free solution, EGTA (added as 1 mM) was used. All solutions were maintained at 30 + 1°C. The rate of stimulation was 1/min. 2.3. Drug
Indapamide* was prepared daily using a reference solution at pH 8. Indapamide was found to be most soluble in alkaline solution since the pKa of the molecule was 8.3 (Campbell et al., 1977). No effect of pH 8 was observed on the amplitude and kinetics of the action potential and contractility. 2.4. Double sucrose gap apparatus
A double sucrose gaP technique has been used to polarize and voltage clamp small strips of mammalian portal vein. The perfusion bath and the electronic apparatus have * Fludex @.
J. MIRONNEAU, Y.M. GARGOUIL been described in detail previously (Rougier et al., 1968). An estimate of the resting potential of the strips was obtained as follows: the preparation in the test gap was perfused with high potassium solution and the electronic setup was connected for current clamp. Then, when the high potassium solution was changed to the reference solution, the preparation repolarized at a stable value. The average gap potential was --45 + 7 mV (mean value +-S.E. of the mean in 30 preparations). Because the short-circuiting factor of the system was a b o u t 0.9 and because the observed gap potential was very close to the potential recorded b y intracellular microelectrodes (Somlyo et al., 1969; Kuriyama et al., 1971), this gap potential was accepted as representing the average resting potential of the cells in the test compartment. Adequate transmembrane voltage control was limited b y the presence of a significant series resistance, and the uniformity of voltage control was limited by the multicellular and inherent cable properties of the muscle strips ( R a m o n et al., 1975). Under our experimental conditions, the test compartment width of 1 0 0 p m was narrow enough for clamping the membrane of resting preparations (space constant of 0.66 mm, Ito and Kuriyama, 1971) and during the active state (membrane resistance is reduced to onetenth, and the space constant to 0.2 mm) so that a spatial uniformity of voltage clamp could be obtained. Direct evidence of the reliability of voltage clamps has been obtained previously on uterine smooth muscle b y measuring the voltage in the test gap independently with a microelectrode (Grosset and Mironneau, 1977). This microelectrode test also indicated that portal vein cells did not escape voltage control when the width of the test gap was a b o u t 100 pm and the current intensity less than 3 pA (fig. 2A). However, the results presented in this paper should be regarded as a comparison between a control and a experimental state (the errors in b o t h states remaining almost indentic~l)
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE
and should be interpreted carefully, as proposed b y Anderson (1977).
59
of differences b e t w e e n the control and test data using a Student's t-test.
2.5. Contraction 3. Results
A photoelectric device was used for measuring the contraction of the muscle in the test c o m p a r t m e n t (Mironneau, 1973). Movem e n t was confined to the part of the muscle in the test c o m p a r t m e n t and a change in the light intensity impinging on the photomultiplier was produced during the contraction. The photoelectric variation was linearly related to the area occluded b y the preparation in a given circular light field (Grosset and Mironneau, 1977).
2.6. Expression of the results The nomenclature used to express the results was as follows: V (mV), variation of the membrane potential, the resting potential being taken as zero. Positive values of V represent a depolarization, negative values a hyperpolarization; I (#A) is the membrane current. Positive values of I correspond to an outward current, negative values to an inward current. Statistics were calculated on the ~ s i s
3.1. Effects o f indapamide on the action potential and contraction o f rabbit portal vein All-or-none action potentials were generally recorded when depolarizing current pulses were applied to the portal vein. Under these conditions, the action potential had an amplitude o f 45_+ 1 0 m V (n = 30) and a duration o f 200 -+ 40 msec (n = 30). They were characterized b y similar rates of depolarization and repolarization; both maximal rates varied between 5 and 10 V/sec (fig. 1A). When an action potential was triggered, a contraction developed. The onset of contraction was delayed, often appearing after 100 or 150 msec, and the total duration was 30-50 sec. Cumulative concentration--response curves for the inhibitory effect of indapamide on the action potential amplitude were obtained in 5 vascular preparations. The results of a typical experiment are shown in fig. lB. The
A
10 mVL A
|
c
0G
1
2
5
10 (xl(~ 4 )
lndapamide (glrnl)
Fig. 1. (A) Simultaneous recordings o f action potential and contraction (in arbitrary units) in longitudinal smooth muscle o f rabbit portal vein. Time base for V and I 50 msec; for C 2 sec. (B) Log concentration--response relationship o f t h e effect o f indapamide (abscissa) on the action potential amplitude (ordinate). At 10 -3 g/ml all o r - n o n e action potentials could not be triggered.
60
J. MIRONNEAU, Y.M. GARGOUIL
Under voltage clamp conditions, the current tracings made for various sustained depolarizations suggested that at least two ionic conductances were involved. Following the
fast and transient surge of capacitive current (whose time constant varied between 3 and 5 msec), there was an inward current (fig. 2A). It was time-and-voltage dependent and was followed by an outward current of large amplitude. The inward current (filled circles) had a low threshold (fig. 2B); it reached its peak amplitude at about + 3 0 mV and reversed at about + 60 mV if the leak current was assumed to be linearly voltage dependent (broken line). The outward current (open circles) showed an outward going rectification, when measured at the end of the pulse. The nature of the ionic current can be demonstrated by studying the effects of permeability inhibitors and of different external ionic concentrations. Manganese (5 mM) or D600 (0.5 mM) suppressed the
A
B
reduction in the amplitude of the action potential was plotted as a function of the logarithm of the external indapamide concentration. The half-maximal response was 1.25 X 10 -4 g/ml. In all experiments, the measurements were made only when the preparations reached a steady state (i.e. after 3--5 min in the test solution). The effects of indapamide were therefore investigated on ionic currents and contraction of rabbit portal vein, at a concentration of 10 -4 g/ml.
3.2. Effects o f indapamide on ionic currents
~A °2,
v,-lc_r
.1
j,
S
S'
-10
.40
•7 0 m V ...L_.._
Vc -0,5.
Fig. 2. (A) Inward (solid circles) and outward (open circles) membrane currents (I) recorded during the application of a rectangular depolarizing pulse (Vc). The transmembrane potential was measured when the microelectrode was inside the cell (Vi). This result shows that there was no important variation between Vc (command voltage) and Vi during the inward or outward current, suggesting that the cells in the test gap remained under adequate voltage control. (B) Current---voltage relationships obtained for the inward (filled circles) and outward (open circles) currents. The reversal potential is close to +60 mV if the leak current is assumed to be linearly voltage dependent (broken line).
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE
inward current in portal vein strips. Similar results were obtained after removing the external calcium ions. These results suggest that the inward current is essentially calciumdependent and are in good agreement with those of Daemers-Lambert (1976). The outward current, recorded for a depolarization at which the calcium current became outward, was greatly decreased after the addition of TEA (20 mM), suggesting potassium dependence. The reversal potential of the outward current, determined by inversion of the tail current at the end of a depolarizing pulse of +60 mV and 200 msec, was --25 + 7.5 (n = 10) referring to the resting potential taken as zero (i.e. --70 mV). The reversal potential was not modified when tested after longer depolarizations. The value of the reversal potential could be considered to be close to the equilibrium potential for potassium ions (--80 to --90 mV) as already calculated by Haljam~ie et al. (1970) and WahlstrSm (1973). Indapamide (10 -4 g/ml) decreased both inward and outward currents. In order to simplify the analysis of the inward current, TEA (20 mM) was added to reference solution to inhibit the outward current which could be mixed with the initial inward current because of its relatively fast activation kinetics. The current-voltage relationships showed that there was no modification of the reversal potential of the calcium current in the presence of indapamide. Furthermore, the reversal potential of the outward current was not modified. The effects of indapamide on the different electrophysiological variables are shown~}l table 1. ReversibIe alterations in the ionic current parameters were always observed under our experimental conditions.
3.3. Effects o f indapamide on contraction Short-lasting depolarizations (50 msec) activating only the calcium inward current were able to trigger a component of contraction which has been called phasic con-
61 TABLE 1 Effects of indapamide on various electrophysiological variables and on contraction (percentage change). Mean value -+ S.E.; in parentheses: number o f preparations. Indapamide (10 -4 g/ml) Action potential amplitude
--35 + 5 3 (11)
Inward calcium current ( maximal intensity)
--27 + 8 1 (9)
Outward potassium current (at +50 mV)
--19 + 4 2
Phasic contraction (maximal contraction)
--29 + 4 3 (7)
Tonic contraction (maximal contraction at +50 mV)
--4 + 3
(9)
(6)
1 p < 0.05;
2 p < 0.01; 3 p < 0.001.
traction (fig. 3A). By replacing the reference solution b y a manganese solution (5 raM), both calcium current and phasic contraction were suppressed. Similar results were obtained in a calcium-free solution. In fig. 3B, the values for the peak contraction are plotted as a function of voltage. Above threshold, the phasic contraction increased to reach a maximal value (+40 mV) then declined. These results seem to support the hypothesis of a fundamental role for the calcium current in the activation of vascular contraction. However, when the duration of a depolarization ( + 5 0 m V ) was increased from 50 to 400 msec, the contraction was enhanced b y a b o u t 30--35% (fig. 4A), suggesting that there was a second phase of increasing tension. This c o m p o n e n t of contraction, called tonic contraction, was recorded in manganese solution or in calcium-free solution for depolarizations longer than 100 msec and greater than +20 mV. The slopes of the contraction and relaxation phases were less steep than those recast/red in manganese-free solution and the maximal amplitude was smaller. The relation between peak tonic contraction and voltage
62
J. MIRONNEAU, Y.M. GARGOUIL
B
A
%Crr~x
50 mVI 80.
VI
b~
a
50ms
(
10,5pA 0
I
0
I
I
l
40
80 rnV
Fig. 3. (A) Effect of a manganese solution on the inward current and on contraction: (a) TEA solution, (b) after the addition of manganese ions (5 raM). (B) Relationship between peak contractions and voltage in response to 50 msec depolarizations. The ordinate is expressed as a percentage of the maximal contraction triggered by an action potential.
has a sigmoid shape (fig. 4B) and indicates that the tonic contraction represented about 20% of the total contraction when a depolarizing pulse similar to the action potential amplitude (+50 mV) and duration (200 msec) was applied to the vascular preparation.
Indapamide decreased the phasic contraction obtained with depolarizations of 50 msec duration (fig. 5A; table 1). When the calcium current was blocked (due to the action of manganese) the tonic contraction was not significantly modified by indapamide (table
A
B 50mVL 100m s
a
%( max 20.
|
0
|"~
40
|
I
8 0 mV
Fig. 4. (A) Effect of two different step durations (a 50 msec; b 400 msec) upon contraction. With a long depolarization, the contraction increased in amplitude and duration. (B) Relationship between peak contractions and voltage in response to 200 msec depolarizations in the presence of manganese (5 raM). The ordinate is expressed as a percentage of the maximal contraction triggered by an action potential.
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE
63
A
B %( max
% C max a
80.
40.
!
0
!
40
I
I~
i
8 0 mV
i
0
I
I
40
8 0 mV
Fig. 5. (A) Effect of indapamide (10 -4 g/ml) on the phasic contraction--voltage relationship: (a) TEA solution, (b) after adding indapamide. (B) No effect of indapamide (filled squares) on the tonic contraction--voltage relationship when compared to manganese solution (filled circles). The ordinates are expressed as a percentage of the maximal contraction triggered by an action potential.
1). The relation between peak contraction and voltage did n o t vary whether indapamide was present or n o t (fig. 5B).
3.4. Influence o f indapamide on noradrenaline-induced contraction The effects of noradrenaline on the portal vein differ with the concentrations used (Holman et al., 1968). At low concentrations (10 -8 g/ml), noradrenaline caused an increase in frequency of the spikes and consequently could induce an increase in contractility. Higher doses (10-6g/ml) caused sustained depolarization and contraction (Godfraind and Kaba, 1972; Golenhofen et al., 1973; Godfraind, 1976). The action of noradrenaline at low concentrations, was studied on the ionic currents and various components of the contraction which develop during an action potential. Noradrenaline (10 -8 g/ml) did not affect either the phasic contraction or the calcium current obtained with 50 msec depolarizations (table 2; fig. 6A). When the inward current was blocked with manganese, both tonic contraction and outward current were increased b y noradrenaline in response to a 200 msec depolarizing step (fig. 6B; table 2). These results show that the stimulation in
contractility induced b y low concentrations of noradrenaline seems to depend essentially on the increase in the tonic contraction, and consequently on the release o f intracellular calcium. Treatment with indapamide (10 -4 g/ml) did not reduce the increase in tonic contraction and outward current due to noradrenaline (fig. 6B; table 2).
A
a
b
~-V
F2~ '
t--
~
B a
50mV
b
c
Fig. 6. (A) No effect of noradrenaline on the inward current and phasic contraction: Ca) TEA solution, (b) after the addition o f noradrenaline (10 -a g/ml). (B) Outward current and tonic contraction in manganese solution Ca), after the addition of noradrenaline (b), and in the presence o f both noradrenaline and indapamide (c). The contraction is expressed as a percentage of the maximal contraction triggered b y an action potential.
64
J. MIRONNEAU, Y.M. GARGOUIL
TABLE 2
TABLE 3
Influence of indapamide on electrophysiological variables and on contractions modified by noradrenaline (percentage change). Mean values -+ S.E.; in parentheses: number of preparations.
Influence of indapamide on electrophysiological variables and on contractions modified by angiotensin II (percentage change).
Outward potassium current (at +50 mV) Tonic contraction (maximal contraction at +50 mV)
Noradrenaline (10 -8 g/ml)
Noradrenaline (10 -s g/ml)+ Indapamide (10 -4 g/ml)
+8_+ 31 (7)
+ 5 + 2 1 (7)
+ 1 5 + 3 2 (7)
+12+_22 (5)
Mean values + S.E.; in parentheses: number of preparations.
Inward calcium current (maximal intensity) Phasic contraction (maximal contraction)
Angiotensin II (10 -7 g/ml)
Angiotensin II (10 -7 g/ml)+ Indapamide (10 -4 g/ml)
+32 + 7 I (9)
--4 + 2 (7)
+34 + 10 1 (9)
--4_+ 3 (6)
1 p < 0.01. 1 p < 0.05;2 p < 0.01.
3.5. Influence o f indapamide on angiotensin II-induced contraction A n g i o t e n s i n I I is k n o w n t o i n d u c e excitat i o n in a w i d e v a r i e t y o f vascular a n d visceral s m o o t h m u s c l e p r e p a r a t i o n s (Regoli et al., 1974; Weston and Golenhofen, 1976; Hamon a n d Worcel, 1 9 7 7 ; S t L o u i s et al., 1 9 7 7 ) . A t a n g i o t e n s i n I I c o n c e n t r a t i o n s in t h e r a n g e o f 1 0 - ~ - - 1 0 -6 g / m l , c o n s i d e r a b l e d e s e n s i t i z a t i o n o c c u r r e d if t h e interval b e t w e e n doses was less t h a n 20 m i n . T h e r e f o r e a t least this a m o u n t o f t i m e was a l l o w e d t o elapse
b e t w e e n successive doses. A t 10 -7 g/ml, a n g i o t e n s i n I I did n o t m o d i f y t h e resting pot e n t i a l b u t increased b o t h i n w a r d c u r r e n t a n d phasic c o n t r a c t i o n (fig. 7). T h e d e l a y e d o u t w a r d c u r r e n t was slightly increased b u t t h e tonic contraction remained unchanged (table
3). I n t h e p r e s e n c e o f a n g i o t e n s i n I I indapam i d e (10 -4 g / m l ) r e d u c e d b o t h i n w a r d c u r r e n t a n d phasic c o n t r a c t i o n t o t h e i r r e f e r e n c e values (fig. 7; t a b l e 3), suggesting t h a t indapamide may inhibit the stimulating effects o f a n g i o t e n s i n I I o n b o t h electrical a n d m e c h a n i c a l a c t i v i t y in p o r t a l vein.
4. D i s c u s s i o n a
v-~
' -f-I
b
•
5Ores
.
c
.
Io,~,--,A-Y'fl._
Fig. 7. Inward calcium current and phasic contraction in TEA solution (a), after the addition of angiotensin II (10 -7 g/ml) (b), and in the presence of both angiotensin II and indapamide (c). The contraction is expressed as a percentage of the maximal contraction triggered by an action potential.
4.1. Excitation--contraction coupling in vascular smooth muscle O u r e x p e r i m e n t s s h o w e d t h a t t h e ionic m e c h a n i s m s in l o n g i t u d i n a l s m o o t h m u s c l e s isolated f r o m t h e p o r t a l vein w e r e v e r y similar t o t h o s e in visceral s m o o t h m u s c l e s (Anderson, 1969; Mironneau, 1973; Kao and McCullough, 1975; Vassort, 1975). Moreover, s i m u l t a n e o u s r e c o r d i n g s o f ionic c u r r e n t s a n d
INDAPAMIDE ON VASCULARSMOOTHMUSCLE contraction suggested that there are at least two types of contraction which differ in their time and voltage dependence. Short-lasting depolarizations (50msec) cause an inward flow of calcium ions which can trigger a phasic component in the contraction. The existence of this phasic contraction is strongly suggested for the following reasons: (a) there is a relationship between the amplitude of the contraction and of the calcium inward current, both being a function of voltage; (b) both contraction and inward current are abolished in calcium-free and manganese~containing solutions. These results are in good agreement with those of Godfraind and Kaba (1972), Collins et al. (1972) and Sigurdsson et al. (1975) with various vascular smooth muscles. In manganese solution or in calcium-free solution, long-lasting depolarizations (100-400 msec) produced a second component in the contraction which was independent of the inward calcium current. The magnitude of this tonic contraction increased with the depolarizations. The presence of this second mechanism is consistent with the hypothesis that calcium can be released from intracellular stores (microvesicles of the surface membrane and sarcoplasmic reticulum) from which it can be displaced by long-lasting depolarizations (Somlyo et al., 1971; McGuffee and Bagby, 1976). During an action potential, the phasic contraction is rapidly activated and represents about 80--90% of the total contraction while the tonic contraction develops slowly and is responsible for 10--20% of the total contraction. All these results support the existence of a dual source of calcium responsible for the activation of contractile proteins in portal vein smooth muscle, as previously described for other smooth muscles (Mironneau, 1973; Godfraind, 1976; Casteels et al., 1977).
4.2. Indapamide dosage In order to explain the electrophysiological effects of indapamide, it is necessary to compare data obtained at similar concentrations.
65 The therapeutic effects of indapamide are obtained with daily doses of 0.5 to 1 mg/kg. The differences between the therapeutic doses and the concentrations used in the present experiments (100 times higher) could be dependent upon: (a) an incomplete solubility of indapamide at pH 8; (b) the short duration of the electrophysiological analysis (3--5rain) as compared to clinical application (2 weeks or more); (c) the noticeable accumulation of indapamide in vascular smooth muscles during therapeutic administration; indapamide can reach a concentration 10 times higher than the plasma concentration (Campbell et al., 1977); (d) the high solubility in lipids suggesting that an important fraction of indapamide can bind to the vaseline seals of the double sucrose gap apparatus; (e) the relative low experimental temperature (30°C). Under these conditions, the concentration of indapamide which really acts at the cellular level could be noticeably lower than the quantity of drug added to the physiological solution. Nevertheless, the concentrations used in the present data have to be considered as pharmacological doses which allow the study of the effects of indapamide in short experiments.
4.3. Electrophysiological effects of indapamide
and
contractile
How indapamide acts on vascular membranes is revealed when voltage clamp data are analyzed. As previously described by Hodgkin and Huxley (1952) the decrease of an ionic current can be related to: (a) a reduction in maximal conductance, or (b) a decrease of the driving force for ion movement (shift of the reversal potential towards less positive voltages). The most obvious effect of indapamide seems to be its ability to inhibit inward and outward currents. The diminution in inward and outward current intensity cannot be related to a variation of the calcium and potassium reversal potentials as these remain stable in the presence of indapamide. Our results tend to show that indapamide
66
essentially acts on calcium and potassium conductances. Indapamide decreased b o t h the calcium inward current and the phasic contraction. This action could explain the depressing effect of indapamide on the contraction of vascular s m o o t h muscles and suggests a antihypertensive property of the drug. Indapamide is n o t able to counterbalance immedia tely the effect of noradrenaline on ionic currents and contraction. This could be due to the fact that noradrenaline also acts b y increasing the tonic contraction thought to be d e p e n d e n t on the release of internal calcium. However, for long-lasting experiments ( 2 0 - - 3 0 m i n ) , indapamide can reduce the amplitude of the noradrenaline-induced contraction. It is suggested that a depletion in the internal stores of calcium could occur due to the sustained inhibition of the inward calcium current b y indapamide. On the other hand, indapamide can rapidly inhibit the stimulating effects of angiotensin II on the inward calcium current and phasic contraction. Moreover, preliminary data indicated that the effect of indapamide on portal vein seemed to be relatively specific since chlortalidone (10 -3 g/ml), a typical diuretic, had no effect on the action potential time course. It can be concluded that indapamide acts primarily on the plasma membrane b y reducing the transmembrane calcium current although a secondary decrease of the intracellular calcium during long exposures to indapamide could not be completely excluded.
Acknowledgements This work was supported by grants from D.G.R.S.T. and I.N.S.E.R.M., France.
References Abe, Y., 1971, Effects of changing the ionic environment on passive and active properties of pregnant rat uterus, J. Physiol. London 214, 173. Anderson, N.C., 1969, Voltage-clamp experiments on uterine smooth muscle, J. Gen. Physiol. 54,145.
J. MIRONNEAU, Y.M. G A R G O U I L Anderson, N.C., 1977, Limitations and possibilities in smooth muscle voltage clamp, in: Excitation .... contraction Coupling in Smooth Muscle, eds. R. Casteels et al. (Elsevier/North Holland Biomed. Press) p. 81. Armstrong, C.M., 1966, Time course of TEA-induced anomalous rectification in squid giant axons, J. Gen. Physiol. 50,491. Beregi, L.G., 1977, Antihypertensive and saluretic properties of the indoline and iso-indoline series, Curt. Med. Res. Opin. 5, S1, 3. Campbell, D.B., A.R. Taylor, X.W. Hopkins and J.R.B. Williams, 1977, Pharmaco-kinetics and metabolism of indapamide: a review, Curt. Med. Res. Opin. 5, $ 1 , 1 3 . Canicave, J.C. and F.X. Lesbre, 1977, Measurement of peripheral resistance b y carotid pulse wave recordings: study of a vasopressor and antihypertensive agent, Curr. Med. Res. Opin. 5, $ 1 , 7 9 . Casteels, R., K. Kitamura, H. Kuriyama and H. Suzuki, 1977, Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery, J. Physiol. Lond. 271, 63. Collins, G.A., M.C. Sutter and J.C. Teiser, 1972, Calcium and contraction in the rabbit mesentericportal vein, Can. J. Physiol. Pharmacol. 50, 289. Daemers-Lambert, C., 1976, Voltage clamp studies on rat portal vein, in: Physiology of smooth muscle, eds. E. Biilbring and M.F. Shuba (Raven Press, New-York) p. 83. Finch, L., P.E. Hicks and R.A. Moore, 1977, The effects of indapamide on vascular reactivity in experimental hypertension, Curr. Med. Res. Opin. 5, S1, 44. Fleckenstein, A., 1977, Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle, Ann. Rev. Pharmacol. Toxicol. 17,149. Godfraind, T., 1976, Calcium exchange in vascular smooth muscle, action of adrenaline and lanthanum, J. Physiol. London 260, 21. Godfraind, T. and K. Kaba, 1972, Blockade or reversal of contraction induced by calcium and adrenaline in depolarized arterial smooth muscle, Brit. J. Pharmacol. 36, 549. Gargouil, Y.M. and J. Mironneau, 1977, Effects of indapamide on excitation-contraction coupling in smooth muscle of the mammalian portal vein, Curr. Med. Res. Opin. 5, $ 1 , 5 5 . Golenhofen, K., N. Hermstein and E. Lammel, 1973, Membrane potential and contraction of vascular smooth muscle (portal vein) during application of noradrenaline and high potassium, and selective inhibitory effect of iproveratril (verapamil), Microvasc. Res. 5, 73. Grosset, A. and J. Mironneau, 1977, An analysis of the actions of prostaglandin E1 on membrane
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE currents and contraction in uterine smooth muscle, J. Physiol. London 270, 765. Haljam~/e, H., B. Johansson, O. Johnsson and H. RScke.rt, 1970, The distribution of sodium, potassium and chloride in the smooth muscle of the rat portal vein, Acta Physiol. Sand. 78, 255. Hamon, G. and M. Worcel, 1977, Mechanism of action of angiotensin II on the membrane potential of rat myometrium, Brit. J. Pharmacol. 51,497 P. Hodgkin, A.L. and A.F. Huxley, 1952, A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. London 117, 500. Holman, M.E., C.B. Kasby, M.B. Suthers and J.A.F. Wilson, 1968, Some properties of the smooth muscle of rabbit portal vein, J. Physiol. London 196, 111. Ito, Y. and H. Kuriyama, 1971, Membrane properties of the smooth muscle fibres of the guinea-pig portal vein, J. Physiol. London 214,427. Kao, C.Y. and J.R. McCullough, 1975, Ionic currents in the uterine smooth muscle, J. Physiol. London 246, 1. Kuriyama, H., K. Oshima and Y. Sakamoto, 1971, The membrane properties of the smooth muscle of the guinea-pig portal vein in isotonic and hypertonic solutions, J. Physiol. London 217, 179. McGuffee, L.J. and R.M. Bagby, 1976, Ultrastructure, calcium accumulation, and contractile response in smooth muscle, Amer. J. Physiol. 230, 1217. Mironneau, J., 1973, Excitation-contraction coupling in voltage-clamped uterine smooth muscle, J. Physiol. London 233,127. Mironneau, J. and J.P. Savineau, 1977, Ionic currents and contraction in vascular smooth muscle, Proc. XXVII Intern. Congr. Physiol. Sci. Paris 13, 515. Ramon, F., N. Anderson, R.W. Joyner and J.W. Moore, 1975, Axon voltage-clamp simulation. IV.
67 A multicellular preparation, Biophys. J. 15, 55. Regoli, D., W.K. Park and F. Rioux, 1974, Pharmacology of angiotensin, Pharmacol. Rev. 26, 69. Rougier, O., G. Vassort and R. St~h-npfli, 1968, Voltage-clamp experiments on frog atrial heart muscle fibres with the sucrose gap technique, Pfliigers Arch. Ges. Physiol. 301, 91. Sigurdsson, S.B., B. Uvelius and B. Johansson, 1975, Relative contribution of superficially bound and extracellular calcium to activation of contraction in isolated rat portal vein, Acta Physiol. Scand. 95, 263. Somlyo, A.V., P. Vinall and A.P. Somlyo, 1969, Excitation--contraction coupling and electrical events in two types of vascular smooth muscle, Microvasc. Res. 1,354. Somlyo, A.P., C.E. Devine, A.V. Somlyo and S.R. North, 1971, Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium-free solutions, J. Cell. Biol. 51, 722. StLouis, J., D. Regoli, J. Barab~ and W.K. Park, 1977, Myotrophic actions of angiotensin and noradrenaline in strips of rabbit aortae, Can. J. Physiol. Pharmacol. 55, 1056. Vassort, G., 1975, Voltage-clamp analysis of transmembrane ionic currents in guinea pig-myometrium: evidence for an initial potassium activation triggered by calcium influx, J. Physiol. London 252, 713. WahlstrSm, B.A., 1973, Ionic fluxes in the rat vortal vein and the applicability of the Goldman equation in predicting the membrane potential from flux data, Acta Physiol. Scand. 89,436. Weston, A.H. and K. Golenhofen, 1976, Comparison of the excitatory and inhibitory effects of angiotensin and vasopressin on mammalian portal vein, Blood Ves. 13,350.
57
ACTION OF INDAPAMIDE ON EXCITATION--CONTRACTION COUPLING IN VASCULAR SMOOTH MUSCLE JEAN MIRONNEAU * and YVES-MICHEL GARGOUIL
Laboratoire de Physiologie Cellulaire, Universit~ de Bordeaux 2, et Laboratoire de Physiologie Animale, Universitg de Poitiers, France Received 24 January 1979, revised MS received 27 March 1979, accepted 4 April 1979
J. MIRONNEAU and Y.M. GARGOUIL, Action o f indapamide on the excitation--contraction coupling in vascular smooth muscle, European J. Pharmacol. 57 (1979)57--67. The effects of indapamide on electrophysiological and mechanical parameters of longitudinal smooth muscle strips isolated from mammalian portal vein were studied by means of a double sucrose gap method associated with a photoelectric device for recording contraction. Indapamide (10 -4 M) reduced both the amplitude of the action potential and the contraction. The calcium inward current decreased and consequently the phasic contraction was also reduced. The potassium outward current was diminished while the tonic contraction was not modified significantly. The depressant nature of the indapamide response could counterbalance the stimulating action of angiotensin II but not that of noradrenaline. The results suggest that indapamide acts primarily on the plasma membrane of vascular smooth muscle by reducing the transmembrane calcium current, although a secondary decrease in the intracellular bound calcium could not be completely excluded. Vascular smooth muscle
Ionic currents
Contraction
1. Introduction Indapamide (chloro-4-N-(methyl-2-indolinyl-1)-sulphamoyl-3-benzamide) is a new drug which possesses antihypertensive and diuretic properties (Beregi, 1977). It has been shown that indapamide decreased peripheral arterial resistance as evaluated by analysing the carotidogram (Canicave and Lesbre, 1977) and measuring blood pressure (Finch et al., 1977); these results suggested a possible effect of indapamide on the contractility of vascular smooth muscle. By using a double sucrose gap device, the electrical properties of smooth muscle cells can be investigated with external electrodes. This method has been applied to several visceral smooth muscles (Anderson, 1969; * Present adress: Dr. J. Mironneau, Laboratoires de Physiologic Cellulaire et de Cytologic, Avenue des Facult~s, 33405 Talence Cedex, France.
Indapamide
Mironneau, 1973; Kao and McCullough, 1975; Vassort, 1975) and, more recently, to vascular smooth muscle (Daemers-Lambert, 1976; Mitonneau and Savineau, 1977). Our experiments were designed to study the relations between contraction, ionic currents and transmembrane potentials in mammalian portal vein. The effects of indapamide on excitation-contraction coupling in portal vein were studied by assuming that the drug may act at the cellular level. A preliminary report of part of this work has already been published (Gargouil and Mironneau, 1977).
2. Materials and methods
2.1. Preparation Isolated longitudinal strips (50--150 #m in diameter, 3--4 mm in length) from the portal vein of rabbits were used in these
58 experiments. After a short healing period (about 15--30 min) in the reference solu. tion, the preparation was ready for current or voltage clamping. 2.2. Solutions
The physiological solutions had the following composition: (a) Reference solution (mM): NaC1 130; KC1 5.6; CaC12 2; MgCI2 0.24; glucose 11. The solution was aerated with 02 and buffered with Tris-HC1 (8.3 mM) at pH 8. (b) The high potassium solution was prepared b y substituting NaC1 for KC1 in equimolar amounts (135,6 raM). (c) The following inhibitors of permeability were used: manganese chloride (5 mM) and D600 (a-isopropyl-a- [ (N-methyl-N-homoveratryl)-~-aminopropyl] -3,4,5-trimethoxyphenylacetonitriteHCI, 0.5 mM) considered to be inhibitors of the calcium inward current (Abe, 1971; Mironneau, 1973; Fleckenstein, 1977) while t e t r a e t h y l a m m o n i u m chloride (20 mM) is k n o w n to inhibit the potassium current (Armstrong, 1966). In order to obtain a calcium-free solution, EGTA (added as 1 mM) was used. All solutions were maintained at 30 + 1°C. The rate of stimulation was 1/min. 2.3. Drug
Indapamide* was prepared daily using a reference solution at pH 8. Indapamide was found to be most soluble in alkaline solution since the pKa of the molecule was 8.3 (Campbell et al., 1977). No effect of pH 8 was observed on the amplitude and kinetics of the action potential and contractility. 2.4. Double sucrose gap apparatus
A double sucrose gaP technique has been used to polarize and voltage clamp small strips of mammalian portal vein. The perfusion bath and the electronic apparatus have * Fludex @.
J. MIRONNEAU, Y.M. GARGOUIL been described in detail previously (Rougier et al., 1968). An estimate of the resting potential of the strips was obtained as follows: the preparation in the test gap was perfused with high potassium solution and the electronic setup was connected for current clamp. Then, when the high potassium solution was changed to the reference solution, the preparation repolarized at a stable value. The average gap potential was --45 + 7 mV (mean value +-S.E. of the mean in 30 preparations). Because the short-circuiting factor of the system was a b o u t 0.9 and because the observed gap potential was very close to the potential recorded b y intracellular microelectrodes (Somlyo et al., 1969; Kuriyama et al., 1971), this gap potential was accepted as representing the average resting potential of the cells in the test compartment. Adequate transmembrane voltage control was limited b y the presence of a significant series resistance, and the uniformity of voltage control was limited by the multicellular and inherent cable properties of the muscle strips ( R a m o n et al., 1975). Under our experimental conditions, the test compartment width of 1 0 0 p m was narrow enough for clamping the membrane of resting preparations (space constant of 0.66 mm, Ito and Kuriyama, 1971) and during the active state (membrane resistance is reduced to onetenth, and the space constant to 0.2 mm) so that a spatial uniformity of voltage clamp could be obtained. Direct evidence of the reliability of voltage clamps has been obtained previously on uterine smooth muscle b y measuring the voltage in the test gap independently with a microelectrode (Grosset and Mironneau, 1977). This microelectrode test also indicated that portal vein cells did not escape voltage control when the width of the test gap was a b o u t 100 pm and the current intensity less than 3 pA (fig. 2A). However, the results presented in this paper should be regarded as a comparison between a control and a experimental state (the errors in b o t h states remaining almost indentic~l)
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE
and should be interpreted carefully, as proposed b y Anderson (1977).
59
of differences b e t w e e n the control and test data using a Student's t-test.
2.5. Contraction 3. Results
A photoelectric device was used for measuring the contraction of the muscle in the test c o m p a r t m e n t (Mironneau, 1973). Movem e n t was confined to the part of the muscle in the test c o m p a r t m e n t and a change in the light intensity impinging on the photomultiplier was produced during the contraction. The photoelectric variation was linearly related to the area occluded b y the preparation in a given circular light field (Grosset and Mironneau, 1977).
2.6. Expression of the results The nomenclature used to express the results was as follows: V (mV), variation of the membrane potential, the resting potential being taken as zero. Positive values of V represent a depolarization, negative values a hyperpolarization; I (#A) is the membrane current. Positive values of I correspond to an outward current, negative values to an inward current. Statistics were calculated on the ~ s i s
3.1. Effects o f indapamide on the action potential and contraction o f rabbit portal vein All-or-none action potentials were generally recorded when depolarizing current pulses were applied to the portal vein. Under these conditions, the action potential had an amplitude o f 45_+ 1 0 m V (n = 30) and a duration o f 200 -+ 40 msec (n = 30). They were characterized b y similar rates of depolarization and repolarization; both maximal rates varied between 5 and 10 V/sec (fig. 1A). When an action potential was triggered, a contraction developed. The onset of contraction was delayed, often appearing after 100 or 150 msec, and the total duration was 30-50 sec. Cumulative concentration--response curves for the inhibitory effect of indapamide on the action potential amplitude were obtained in 5 vascular preparations. The results of a typical experiment are shown in fig. lB. The
A
10 mVL A
|
c
0G
1
2
5
10 (xl(~ 4 )
lndapamide (glrnl)
Fig. 1. (A) Simultaneous recordings o f action potential and contraction (in arbitrary units) in longitudinal smooth muscle o f rabbit portal vein. Time base for V and I 50 msec; for C 2 sec. (B) Log concentration--response relationship o f t h e effect o f indapamide (abscissa) on the action potential amplitude (ordinate). At 10 -3 g/ml all o r - n o n e action potentials could not be triggered.
60
J. MIRONNEAU, Y.M. GARGOUIL
Under voltage clamp conditions, the current tracings made for various sustained depolarizations suggested that at least two ionic conductances were involved. Following the
fast and transient surge of capacitive current (whose time constant varied between 3 and 5 msec), there was an inward current (fig. 2A). It was time-and-voltage dependent and was followed by an outward current of large amplitude. The inward current (filled circles) had a low threshold (fig. 2B); it reached its peak amplitude at about + 3 0 mV and reversed at about + 60 mV if the leak current was assumed to be linearly voltage dependent (broken line). The outward current (open circles) showed an outward going rectification, when measured at the end of the pulse. The nature of the ionic current can be demonstrated by studying the effects of permeability inhibitors and of different external ionic concentrations. Manganese (5 mM) or D600 (0.5 mM) suppressed the
A
B
reduction in the amplitude of the action potential was plotted as a function of the logarithm of the external indapamide concentration. The half-maximal response was 1.25 X 10 -4 g/ml. In all experiments, the measurements were made only when the preparations reached a steady state (i.e. after 3--5 min in the test solution). The effects of indapamide were therefore investigated on ionic currents and contraction of rabbit portal vein, at a concentration of 10 -4 g/ml.
3.2. Effects o f indapamide on ionic currents
~A °2,
v,-lc_r
.1
j,
S
S'
-10
.40
•7 0 m V ...L_.._
Vc -0,5.
Fig. 2. (A) Inward (solid circles) and outward (open circles) membrane currents (I) recorded during the application of a rectangular depolarizing pulse (Vc). The transmembrane potential was measured when the microelectrode was inside the cell (Vi). This result shows that there was no important variation between Vc (command voltage) and Vi during the inward or outward current, suggesting that the cells in the test gap remained under adequate voltage control. (B) Current---voltage relationships obtained for the inward (filled circles) and outward (open circles) currents. The reversal potential is close to +60 mV if the leak current is assumed to be linearly voltage dependent (broken line).
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE
inward current in portal vein strips. Similar results were obtained after removing the external calcium ions. These results suggest that the inward current is essentially calciumdependent and are in good agreement with those of Daemers-Lambert (1976). The outward current, recorded for a depolarization at which the calcium current became outward, was greatly decreased after the addition of TEA (20 mM), suggesting potassium dependence. The reversal potential of the outward current, determined by inversion of the tail current at the end of a depolarizing pulse of +60 mV and 200 msec, was --25 + 7.5 (n = 10) referring to the resting potential taken as zero (i.e. --70 mV). The reversal potential was not modified when tested after longer depolarizations. The value of the reversal potential could be considered to be close to the equilibrium potential for potassium ions (--80 to --90 mV) as already calculated by Haljam~ie et al. (1970) and WahlstrSm (1973). Indapamide (10 -4 g/ml) decreased both inward and outward currents. In order to simplify the analysis of the inward current, TEA (20 mM) was added to reference solution to inhibit the outward current which could be mixed with the initial inward current because of its relatively fast activation kinetics. The current-voltage relationships showed that there was no modification of the reversal potential of the calcium current in the presence of indapamide. Furthermore, the reversal potential of the outward current was not modified. The effects of indapamide on the different electrophysiological variables are shown~}l table 1. ReversibIe alterations in the ionic current parameters were always observed under our experimental conditions.
3.3. Effects o f indapamide on contraction Short-lasting depolarizations (50 msec) activating only the calcium inward current were able to trigger a component of contraction which has been called phasic con-
61 TABLE 1 Effects of indapamide on various electrophysiological variables and on contraction (percentage change). Mean value -+ S.E.; in parentheses: number o f preparations. Indapamide (10 -4 g/ml) Action potential amplitude
--35 + 5 3 (11)
Inward calcium current ( maximal intensity)
--27 + 8 1 (9)
Outward potassium current (at +50 mV)
--19 + 4 2
Phasic contraction (maximal contraction)
--29 + 4 3 (7)
Tonic contraction (maximal contraction at +50 mV)
--4 + 3
(9)
(6)
1 p < 0.05;
2 p < 0.01; 3 p < 0.001.
traction (fig. 3A). By replacing the reference solution b y a manganese solution (5 raM), both calcium current and phasic contraction were suppressed. Similar results were obtained in a calcium-free solution. In fig. 3B, the values for the peak contraction are plotted as a function of voltage. Above threshold, the phasic contraction increased to reach a maximal value (+40 mV) then declined. These results seem to support the hypothesis of a fundamental role for the calcium current in the activation of vascular contraction. However, when the duration of a depolarization ( + 5 0 m V ) was increased from 50 to 400 msec, the contraction was enhanced b y a b o u t 30--35% (fig. 4A), suggesting that there was a second phase of increasing tension. This c o m p o n e n t of contraction, called tonic contraction, was recorded in manganese solution or in calcium-free solution for depolarizations longer than 100 msec and greater than +20 mV. The slopes of the contraction and relaxation phases were less steep than those recast/red in manganese-free solution and the maximal amplitude was smaller. The relation between peak tonic contraction and voltage
62
J. MIRONNEAU, Y.M. GARGOUIL
B
A
%Crr~x
50 mVI 80.
VI
b~
a
50ms
(
10,5pA 0
I
0
I
I
l
40
80 rnV
Fig. 3. (A) Effect of a manganese solution on the inward current and on contraction: (a) TEA solution, (b) after the addition of manganese ions (5 raM). (B) Relationship between peak contractions and voltage in response to 50 msec depolarizations. The ordinate is expressed as a percentage of the maximal contraction triggered by an action potential.
has a sigmoid shape (fig. 4B) and indicates that the tonic contraction represented about 20% of the total contraction when a depolarizing pulse similar to the action potential amplitude (+50 mV) and duration (200 msec) was applied to the vascular preparation.
Indapamide decreased the phasic contraction obtained with depolarizations of 50 msec duration (fig. 5A; table 1). When the calcium current was blocked (due to the action of manganese) the tonic contraction was not significantly modified by indapamide (table
A
B 50mVL 100m s
a
%( max 20.
|
0
|"~
40
|
I
8 0 mV
Fig. 4. (A) Effect of two different step durations (a 50 msec; b 400 msec) upon contraction. With a long depolarization, the contraction increased in amplitude and duration. (B) Relationship between peak contractions and voltage in response to 200 msec depolarizations in the presence of manganese (5 raM). The ordinate is expressed as a percentage of the maximal contraction triggered by an action potential.
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE
63
A
B %( max
% C max a
80.
40.
!
0
!
40
I
I~
i
8 0 mV
i
0
I
I
40
8 0 mV
Fig. 5. (A) Effect of indapamide (10 -4 g/ml) on the phasic contraction--voltage relationship: (a) TEA solution, (b) after adding indapamide. (B) No effect of indapamide (filled squares) on the tonic contraction--voltage relationship when compared to manganese solution (filled circles). The ordinates are expressed as a percentage of the maximal contraction triggered by an action potential.
1). The relation between peak contraction and voltage did n o t vary whether indapamide was present or n o t (fig. 5B).
3.4. Influence o f indapamide on noradrenaline-induced contraction The effects of noradrenaline on the portal vein differ with the concentrations used (Holman et al., 1968). At low concentrations (10 -8 g/ml), noradrenaline caused an increase in frequency of the spikes and consequently could induce an increase in contractility. Higher doses (10-6g/ml) caused sustained depolarization and contraction (Godfraind and Kaba, 1972; Golenhofen et al., 1973; Godfraind, 1976). The action of noradrenaline at low concentrations, was studied on the ionic currents and various components of the contraction which develop during an action potential. Noradrenaline (10 -8 g/ml) did not affect either the phasic contraction or the calcium current obtained with 50 msec depolarizations (table 2; fig. 6A). When the inward current was blocked with manganese, both tonic contraction and outward current were increased b y noradrenaline in response to a 200 msec depolarizing step (fig. 6B; table 2). These results show that the stimulation in
contractility induced b y low concentrations of noradrenaline seems to depend essentially on the increase in the tonic contraction, and consequently on the release o f intracellular calcium. Treatment with indapamide (10 -4 g/ml) did not reduce the increase in tonic contraction and outward current due to noradrenaline (fig. 6B; table 2).
A
a
b
~-V
F2~ '
t--
~
B a
50mV
b
c
Fig. 6. (A) No effect of noradrenaline on the inward current and phasic contraction: Ca) TEA solution, (b) after the addition o f noradrenaline (10 -a g/ml). (B) Outward current and tonic contraction in manganese solution Ca), after the addition of noradrenaline (b), and in the presence o f both noradrenaline and indapamide (c). The contraction is expressed as a percentage of the maximal contraction triggered b y an action potential.
64
J. MIRONNEAU, Y.M. GARGOUIL
TABLE 2
TABLE 3
Influence of indapamide on electrophysiological variables and on contractions modified by noradrenaline (percentage change). Mean values -+ S.E.; in parentheses: number of preparations.
Influence of indapamide on electrophysiological variables and on contractions modified by angiotensin II (percentage change).
Outward potassium current (at +50 mV) Tonic contraction (maximal contraction at +50 mV)
Noradrenaline (10 -8 g/ml)
Noradrenaline (10 -s g/ml)+ Indapamide (10 -4 g/ml)
+8_+ 31 (7)
+ 5 + 2 1 (7)
+ 1 5 + 3 2 (7)
+12+_22 (5)
Mean values + S.E.; in parentheses: number of preparations.
Inward calcium current (maximal intensity) Phasic contraction (maximal contraction)
Angiotensin II (10 -7 g/ml)
Angiotensin II (10 -7 g/ml)+ Indapamide (10 -4 g/ml)
+32 + 7 I (9)
--4 + 2 (7)
+34 + 10 1 (9)
--4_+ 3 (6)
1 p < 0.01. 1 p < 0.05;2 p < 0.01.
3.5. Influence o f indapamide on angiotensin II-induced contraction A n g i o t e n s i n I I is k n o w n t o i n d u c e excitat i o n in a w i d e v a r i e t y o f vascular a n d visceral s m o o t h m u s c l e p r e p a r a t i o n s (Regoli et al., 1974; Weston and Golenhofen, 1976; Hamon a n d Worcel, 1 9 7 7 ; S t L o u i s et al., 1 9 7 7 ) . A t a n g i o t e n s i n I I c o n c e n t r a t i o n s in t h e r a n g e o f 1 0 - ~ - - 1 0 -6 g / m l , c o n s i d e r a b l e d e s e n s i t i z a t i o n o c c u r r e d if t h e interval b e t w e e n doses was less t h a n 20 m i n . T h e r e f o r e a t least this a m o u n t o f t i m e was a l l o w e d t o elapse
b e t w e e n successive doses. A t 10 -7 g/ml, a n g i o t e n s i n I I did n o t m o d i f y t h e resting pot e n t i a l b u t increased b o t h i n w a r d c u r r e n t a n d phasic c o n t r a c t i o n (fig. 7). T h e d e l a y e d o u t w a r d c u r r e n t was slightly increased b u t t h e tonic contraction remained unchanged (table
3). I n t h e p r e s e n c e o f a n g i o t e n s i n I I indapam i d e (10 -4 g / m l ) r e d u c e d b o t h i n w a r d c u r r e n t a n d phasic c o n t r a c t i o n t o t h e i r r e f e r e n c e values (fig. 7; t a b l e 3), suggesting t h a t indapamide may inhibit the stimulating effects o f a n g i o t e n s i n I I o n b o t h electrical a n d m e c h a n i c a l a c t i v i t y in p o r t a l vein.
4. D i s c u s s i o n a
v-~
' -f-I
b
•
5Ores
.
c
.
Io,~,--,A-Y'fl._
Fig. 7. Inward calcium current and phasic contraction in TEA solution (a), after the addition of angiotensin II (10 -7 g/ml) (b), and in the presence of both angiotensin II and indapamide (c). The contraction is expressed as a percentage of the maximal contraction triggered by an action potential.
4.1. Excitation--contraction coupling in vascular smooth muscle O u r e x p e r i m e n t s s h o w e d t h a t t h e ionic m e c h a n i s m s in l o n g i t u d i n a l s m o o t h m u s c l e s isolated f r o m t h e p o r t a l vein w e r e v e r y similar t o t h o s e in visceral s m o o t h m u s c l e s (Anderson, 1969; Mironneau, 1973; Kao and McCullough, 1975; Vassort, 1975). Moreover, s i m u l t a n e o u s r e c o r d i n g s o f ionic c u r r e n t s a n d
INDAPAMIDE ON VASCULARSMOOTHMUSCLE contraction suggested that there are at least two types of contraction which differ in their time and voltage dependence. Short-lasting depolarizations (50msec) cause an inward flow of calcium ions which can trigger a phasic component in the contraction. The existence of this phasic contraction is strongly suggested for the following reasons: (a) there is a relationship between the amplitude of the contraction and of the calcium inward current, both being a function of voltage; (b) both contraction and inward current are abolished in calcium-free and manganese~containing solutions. These results are in good agreement with those of Godfraind and Kaba (1972), Collins et al. (1972) and Sigurdsson et al. (1975) with various vascular smooth muscles. In manganese solution or in calcium-free solution, long-lasting depolarizations (100-400 msec) produced a second component in the contraction which was independent of the inward calcium current. The magnitude of this tonic contraction increased with the depolarizations. The presence of this second mechanism is consistent with the hypothesis that calcium can be released from intracellular stores (microvesicles of the surface membrane and sarcoplasmic reticulum) from which it can be displaced by long-lasting depolarizations (Somlyo et al., 1971; McGuffee and Bagby, 1976). During an action potential, the phasic contraction is rapidly activated and represents about 80--90% of the total contraction while the tonic contraction develops slowly and is responsible for 10--20% of the total contraction. All these results support the existence of a dual source of calcium responsible for the activation of contractile proteins in portal vein smooth muscle, as previously described for other smooth muscles (Mironneau, 1973; Godfraind, 1976; Casteels et al., 1977).
4.2. Indapamide dosage In order to explain the electrophysiological effects of indapamide, it is necessary to compare data obtained at similar concentrations.
65 The therapeutic effects of indapamide are obtained with daily doses of 0.5 to 1 mg/kg. The differences between the therapeutic doses and the concentrations used in the present experiments (100 times higher) could be dependent upon: (a) an incomplete solubility of indapamide at pH 8; (b) the short duration of the electrophysiological analysis (3--5rain) as compared to clinical application (2 weeks or more); (c) the noticeable accumulation of indapamide in vascular smooth muscles during therapeutic administration; indapamide can reach a concentration 10 times higher than the plasma concentration (Campbell et al., 1977); (d) the high solubility in lipids suggesting that an important fraction of indapamide can bind to the vaseline seals of the double sucrose gap apparatus; (e) the relative low experimental temperature (30°C). Under these conditions, the concentration of indapamide which really acts at the cellular level could be noticeably lower than the quantity of drug added to the physiological solution. Nevertheless, the concentrations used in the present data have to be considered as pharmacological doses which allow the study of the effects of indapamide in short experiments.
4.3. Electrophysiological effects of indapamide
and
contractile
How indapamide acts on vascular membranes is revealed when voltage clamp data are analyzed. As previously described by Hodgkin and Huxley (1952) the decrease of an ionic current can be related to: (a) a reduction in maximal conductance, or (b) a decrease of the driving force for ion movement (shift of the reversal potential towards less positive voltages). The most obvious effect of indapamide seems to be its ability to inhibit inward and outward currents. The diminution in inward and outward current intensity cannot be related to a variation of the calcium and potassium reversal potentials as these remain stable in the presence of indapamide. Our results tend to show that indapamide
66
essentially acts on calcium and potassium conductances. Indapamide decreased b o t h the calcium inward current and the phasic contraction. This action could explain the depressing effect of indapamide on the contraction of vascular s m o o t h muscles and suggests a antihypertensive property of the drug. Indapamide is n o t able to counterbalance immedia tely the effect of noradrenaline on ionic currents and contraction. This could be due to the fact that noradrenaline also acts b y increasing the tonic contraction thought to be d e p e n d e n t on the release of internal calcium. However, for long-lasting experiments ( 2 0 - - 3 0 m i n ) , indapamide can reduce the amplitude of the noradrenaline-induced contraction. It is suggested that a depletion in the internal stores of calcium could occur due to the sustained inhibition of the inward calcium current b y indapamide. On the other hand, indapamide can rapidly inhibit the stimulating effects of angiotensin II on the inward calcium current and phasic contraction. Moreover, preliminary data indicated that the effect of indapamide on portal vein seemed to be relatively specific since chlortalidone (10 -3 g/ml), a typical diuretic, had no effect on the action potential time course. It can be concluded that indapamide acts primarily on the plasma membrane b y reducing the transmembrane calcium current although a secondary decrease of the intracellular calcium during long exposures to indapamide could not be completely excluded.
Acknowledgements This work was supported by grants from D.G.R.S.T. and I.N.S.E.R.M., France.
References Abe, Y., 1971, Effects of changing the ionic environment on passive and active properties of pregnant rat uterus, J. Physiol. London 214, 173. Anderson, N.C., 1969, Voltage-clamp experiments on uterine smooth muscle, J. Gen. Physiol. 54,145.
J. MIRONNEAU, Y.M. G A R G O U I L Anderson, N.C., 1977, Limitations and possibilities in smooth muscle voltage clamp, in: Excitation .... contraction Coupling in Smooth Muscle, eds. R. Casteels et al. (Elsevier/North Holland Biomed. Press) p. 81. Armstrong, C.M., 1966, Time course of TEA-induced anomalous rectification in squid giant axons, J. Gen. Physiol. 50,491. Beregi, L.G., 1977, Antihypertensive and saluretic properties of the indoline and iso-indoline series, Curt. Med. Res. Opin. 5, S1, 3. Campbell, D.B., A.R. Taylor, X.W. Hopkins and J.R.B. Williams, 1977, Pharmaco-kinetics and metabolism of indapamide: a review, Curt. Med. Res. Opin. 5, $ 1 , 1 3 . Canicave, J.C. and F.X. Lesbre, 1977, Measurement of peripheral resistance b y carotid pulse wave recordings: study of a vasopressor and antihypertensive agent, Curr. Med. Res. Opin. 5, $ 1 , 7 9 . Casteels, R., K. Kitamura, H. Kuriyama and H. Suzuki, 1977, Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery, J. Physiol. Lond. 271, 63. Collins, G.A., M.C. Sutter and J.C. Teiser, 1972, Calcium and contraction in the rabbit mesentericportal vein, Can. J. Physiol. Pharmacol. 50, 289. Daemers-Lambert, C., 1976, Voltage clamp studies on rat portal vein, in: Physiology of smooth muscle, eds. E. Biilbring and M.F. Shuba (Raven Press, New-York) p. 83. Finch, L., P.E. Hicks and R.A. Moore, 1977, The effects of indapamide on vascular reactivity in experimental hypertension, Curr. Med. Res. Opin. 5, S1, 44. Fleckenstein, A., 1977, Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle, Ann. Rev. Pharmacol. Toxicol. 17,149. Godfraind, T., 1976, Calcium exchange in vascular smooth muscle, action of adrenaline and lanthanum, J. Physiol. London 260, 21. Godfraind, T. and K. Kaba, 1972, Blockade or reversal of contraction induced by calcium and adrenaline in depolarized arterial smooth muscle, Brit. J. Pharmacol. 36, 549. Gargouil, Y.M. and J. Mironneau, 1977, Effects of indapamide on excitation-contraction coupling in smooth muscle of the mammalian portal vein, Curr. Med. Res. Opin. 5, $ 1 , 5 5 . Golenhofen, K., N. Hermstein and E. Lammel, 1973, Membrane potential and contraction of vascular smooth muscle (portal vein) during application of noradrenaline and high potassium, and selective inhibitory effect of iproveratril (verapamil), Microvasc. Res. 5, 73. Grosset, A. and J. Mironneau, 1977, An analysis of the actions of prostaglandin E1 on membrane
INDAPAMIDE ON VASCULAR SMOOTH MUSCLE currents and contraction in uterine smooth muscle, J. Physiol. London 270, 765. Haljam~/e, H., B. Johansson, O. Johnsson and H. RScke.rt, 1970, The distribution of sodium, potassium and chloride in the smooth muscle of the rat portal vein, Acta Physiol. Sand. 78, 255. Hamon, G. and M. Worcel, 1977, Mechanism of action of angiotensin II on the membrane potential of rat myometrium, Brit. J. Pharmacol. 51,497 P. Hodgkin, A.L. and A.F. Huxley, 1952, A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. London 117, 500. Holman, M.E., C.B. Kasby, M.B. Suthers and J.A.F. Wilson, 1968, Some properties of the smooth muscle of rabbit portal vein, J. Physiol. London 196, 111. Ito, Y. and H. Kuriyama, 1971, Membrane properties of the smooth muscle fibres of the guinea-pig portal vein, J. Physiol. London 214,427. Kao, C.Y. and J.R. McCullough, 1975, Ionic currents in the uterine smooth muscle, J. Physiol. London 246, 1. Kuriyama, H., K. Oshima and Y. Sakamoto, 1971, The membrane properties of the smooth muscle of the guinea-pig portal vein in isotonic and hypertonic solutions, J. Physiol. London 217, 179. McGuffee, L.J. and R.M. Bagby, 1976, Ultrastructure, calcium accumulation, and contractile response in smooth muscle, Amer. J. Physiol. 230, 1217. Mironneau, J., 1973, Excitation-contraction coupling in voltage-clamped uterine smooth muscle, J. Physiol. London 233,127. Mironneau, J. and J.P. Savineau, 1977, Ionic currents and contraction in vascular smooth muscle, Proc. XXVII Intern. Congr. Physiol. Sci. Paris 13, 515. Ramon, F., N. Anderson, R.W. Joyner and J.W. Moore, 1975, Axon voltage-clamp simulation. IV.
67 A multicellular preparation, Biophys. J. 15, 55. Regoli, D., W.K. Park and F. Rioux, 1974, Pharmacology of angiotensin, Pharmacol. Rev. 26, 69. Rougier, O., G. Vassort and R. St~h-npfli, 1968, Voltage-clamp experiments on frog atrial heart muscle fibres with the sucrose gap technique, Pfliigers Arch. Ges. Physiol. 301, 91. Sigurdsson, S.B., B. Uvelius and B. Johansson, 1975, Relative contribution of superficially bound and extracellular calcium to activation of contraction in isolated rat portal vein, Acta Physiol. Scand. 95, 263. Somlyo, A.V., P. Vinall and A.P. Somlyo, 1969, Excitation--contraction coupling and electrical events in two types of vascular smooth muscle, Microvasc. Res. 1,354. Somlyo, A.P., C.E. Devine, A.V. Somlyo and S.R. North, 1971, Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium-free solutions, J. Cell. Biol. 51, 722. StLouis, J., D. Regoli, J. Barab~ and W.K. Park, 1977, Myotrophic actions of angiotensin and noradrenaline in strips of rabbit aortae, Can. J. Physiol. Pharmacol. 55, 1056. Vassort, G., 1975, Voltage-clamp analysis of transmembrane ionic currents in guinea pig-myometrium: evidence for an initial potassium activation triggered by calcium influx, J. Physiol. London 252, 713. WahlstrSm, B.A., 1973, Ionic fluxes in the rat vortal vein and the applicability of the Goldman equation in predicting the membrane potential from flux data, Acta Physiol. Scand. 89,436. Weston, A.H. and K. Golenhofen, 1976, Comparison of the excitatory and inhibitory effects of angiotensin and vasopressin on mammalian portal vein, Blood Ves. 13,350.
