J. Physiol. (1977), 271, pp. 63-79 With 11 text-f guree Printed in Great Britain
63
EXCITATION-CONTRACTION COUPLING IN THE SMOOTH MUSCLE CELLS OF THE RABBIT MAIN PULMONARY ARTERY
BY R. CASTEELS, K. KITAMURA,* H. KURIYAMA* AND H. SUZUKI* From the Laboratorium voor Fysiologie Universiteit Leuven, B-3000 Leuven, Belgium (Received 6 December 1976) SUMMARY
1. Increasing the external K concentration depolarizes the smooth muscle cells of the main pulmonary artery, and this depolarization reaches a maximal slope of 58 mV for a tenfold change of [K].. The threshold depolarization for inducing contraction is at 4 mV and the maximal contraction is reached at a [K]o of 58 mM. 2. Noradrenaline concentrations between 2 x 10-8 M and 10-7 M induce tension without depolarizing the cells, but at higher concentrations noradrenaline not only elicits a large tension response but also depolarizes the cells in a dose-dependent way. 3. The effect of noradrenaline on the pulmonary artery is appreciably modified by substituting sucrose for NaCl: the cells are slightly hyperpolarized and the tension response is very much reduced. 4. By studying the tension response to noradrenaline in other experimental conditions which cause a small hyperpolarization of the cells, such as 5 mM-[Ca]o, 2.9 mM-[K]o or a small depolarization, such as 11-9 mM-[K]0, it was found that a slight modification of the membrane potential can exert an important effect on the noradrenaline response. 5. A simultaneous decrease of [Ca]0 and [Na]o reduces the tension response to all noradrenaline concentrations. It was found that a reduction of [Na]o exerts a more depressing effect than a reduction of [Ca]o. In interpreting these results we have to take into account changes of the membrane potential, of availability of Ca, and some competition between external Ca and Na. 6. A study of the effect of different concentrations of noradrenaline in Krebs solutions and Ca-free solution has shown that concentrations up to *
Present address: Department of Physiology, Faculty of Dentistry,
Kyushu
University, Fukuoka 812, Japan.
3
P HY 27I
R. CA STEELS AND OTHERS 2*5 x 10-7 M elicit contraction by increasing the Ca influx, while higher concentrations also induce a release of cellular Ca. 7. Caffeine depolarizes the cells and reduces the membrane resistance. It modifies the K, Cl and Ca fluxes in the same way as noradrenaline, but it suppresses the mechanical response induced by noradrenaline. 64
INTRODUCTION
Su, Bevan & Ursillo (1964) have described that noradrenaline induces a contraction in the smooth muscle cells of the main pulmonary artery without depolarizing the cells or eliciting action potentials. In a later study of the effect of noradrenaline on the membrane potential of these cells, Somlyo & Somlyo (1968) have observed (with the sucrose-gap method) that this substance depolarizes and induces oscillations of the membrane. However, because there was only a limited quantitative correlation between the tension development and the depolarization, and because drug-induced contractions could also occur in depolarized tissues, these authors have called this type of excitation-contraction coupling, pharmacomechanical coupling. In addition, the smooth muscle cells of the pulmonary artery can be activated also by depolarizing the cells with K-rich solutions. Ill the present work we have investigated the effect of different concentrations of noradrenaline and of external K on the tension development and on the membrane potential. We have tried to elucidate the role of changes of the membrane potential in the activation of the contractile proteins and to determine the relative importance of external and cellular Ca in eliciting the mechanical response under various experimental conditions. METHODS
The dissections of the strips of the main pulmonary artery and the measurement of the electrical properties of single cells and of K, Cl and Na fluxes have been described in the preceding paper (Casteels, Kitamura, Kuriyama & Suzuki, 1977). The Ca efflux experiments were performed in Krebs solution containing 1*5 mmCa2+, after a loading period in the radioactive solution of 2 hr. The uptake of 4"Ca by the cellular compartment was estimated by using a modification of the La method described by van Breemen, Farinas, Casteels, Gerba, Wuytack & Deth (1973). After the loading procedure, the tissues were first washed for 5 min in an isotonic Ca-free solution containing 10 m -La. The further efflux was then performed in the same solution, but added with 0.1 mm DNP and 1 mm iodo-acetic acid. This procedure induces a slowing down of the efflux of 4"Ca from the cellular compartment and therefore facilitates the estimation of the amount of 4"Ca in the cellular compartment by extrapolating the slow phase of the efflux to zero time. In order to record simultaneously the electrical and the mechanical activity, we have used the double sucrose-gap method as described by Kuriyama & Tomita (1970). The tension development of the tissues was measured under isometric conditions
EXCITATION-CONTRACTION COUPLING
65 in an organ bath of 3 ml. which was perfused continuously at a rate of 6 ml./min. The temperature was maintained at 35 'C. Both ends of the preparation were tied by cotton strings between a tension transducer and a hook at the bottom of the bath. The composition of the physiological solution, and of the modified solutions, have been described (Casteels et al. 1977). The drugs used in the experiments were noradrenaline HCl, caffeine, EGTA (ethylene glycol bis (fl-aminoethyl ether)-N-N'tetraacetic acid) and ouabain (g-strophantin). The concentrations used in the experiments are given in the Results. RESULTS
Contraction induced by change of [K]o Action potentials normally, are not generated in smooth muscle cells of the pulmonary artery and the mechanical response is induced in these cells either by a depolarization of the membrane or by pharmacomechanical (o)
0I(x)
-10 F E C 0 0
a)C
1
-20 F
C
-30 F
C
-40 F
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a) a)
c
Ea)
-50 F -60
x
0 I
0 1
2
[K]o
I
I
50 5 20 10 concentration (mM)
100
200
Fig. 1. The changes of the membrane potential measured with intracellular electrodes in mV (0) and of the mechanical response (x) induced by different concentrations [K]0, are represented as a function of the log of [K]O. The tension development of the different [K]o is given as a relative value of the tension developed in 137 mM-[K]0.
coupling, which can occur with or without depolarization. In order to investigate the effect of the membrane potential on the mechanical response, we have studied the action of various concentrations [K]0 on the 3-2
66 R. CJASTEELS AND OTHERS membrane potential and tension development. Theseresults arerepresented in Fig. 1. The minimal depolarization to evoke contraction by increasing [K]0 is 4 mV and such a depolarization is obtained at a [K]o of 9 mm. A further increase of [K]o enlarges the amplitude of the tension development, reaching its max. at 58 mm. The graph representing the membrane potential as a function of the logarithm of [K]o has a maximal slope of 58 mV per tenfold change of [K]o. The cells slightly hyperpolarize by
U..fI200 10
mg
1 see 0
200
0 0
.~~~~~~~~0
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S
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~ ~
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o
_
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00*
xa.;~~~0* -45
X0X
X
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-------
--
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-35 -25 Membrane potential (mV)
-15
Fig. 2. Relationship between the membrane potential (mV) and the mechanical response (mg) measured with the double sucrose-gap method. The membrane potential was displaced by application of outward current of various intensities. The membrane potential of three different preparations is represented in the graph by different symbols (0O 0, x ). The upper traces show the actual potential changes and the mechanical responses produced by three different intensities of the outward current pulses.
reducing [K]o to 3 mm, while a further reduction of [K]o results again in a depolarization by some mV and produces a small tension development. Using the double sucrose-gap method we have also investigated the relationship between depolarization of the membrane induced by current injection and the mechanical response (Fig. 2). The membrane potential measured by this method in three different preparations was - 48, -47
EXCITATION-CONTRACTION COUPLING 67 and -42 mV. The lower value of these measurements as compared with the ones obtained with micro-electrodes is due presumably to leakage of current. The depolarization which had to be induced in these preparations to produce contraction were 4, 6 and 3 mV, respectively. We can assume, therefore, that the critical depolarization for inducing contraction is about 4 mV, as suggested already by the experiments with excess [K]o.
-45
2
-50 T
E
Na-defic entA
~0
2.5 x10-6 2-5x10-7 5X10-6 5x10-7 Noradrenaline (M) Fig. 3. Effects of various concentrations of noradrenaline between 2-5 x 10-8 and 5 x 10-6 M on the membrane potential and tension development in Krebs solution and in Na-deficient solution prepared with sucrose. The membrane potential (mV) measured with intracellular electrodes, is given as mean values + S.D. of observatoins of 11-18 penetrations in Krebs solution (0) and in Na-deficient solution (e). The mechanical response is represented by A for the values obtained in Krebs solution and by A for values in Na-deficient solution. Each point represents the mean of seven experiments. 2.5x10-8 Control 5x10-8 Na-deficient
Contraction induced by noradrenaline in Krebs solution We have investigated the effect of different concentrations of noradrenaline between 5 x 10-9 and 5 x 106 M on the membrane potential and tension development of the pulmonary artery. Concentrations of noradrenaline between 2 x 108 M and 10-7 M induce a tension development of the pulmonary artery without affecting the membrane potential. Higher concentrations of noradrenaline cause a progressively increasing tension development, but also depolarize the cells as represented in Fig. 3.
R. CASTEELS AND OTHERS 68 At 5 x 106 M this depolarization reaches a value of 9 mV. This depolarizing action of high concentrations of noradrenaline is due probably to an increase of the permeability of the membrane for Na and Cl, while the concomitant reduction of the membrane resistance depends on the increase of the permeability of the membrane for Na, C1 andK (Casteels et al. 1977). It was found worthwhile therefore, to study the effect of different concentrations of noradrenaline in Na- and Cl-deficient medium, prepared by substituting sucrose for NaCl. This solution only slightly hyperpolarizes the cells, but changes appreciably the effect of noradrenaline on the membrane potential and tension development. An increase of the noradrenaline concentration slightly augments the hyperpolarization of the cells and causes a tension development which remains much smaller over the whole range of the noradrenaline concentration. In addition, the threshold concentration for inducing contraction is shifted up to 2-5 x 10-7 M-
noradrenaline.
Interaction of [Ca]o and noradrenaline in inducing contraction Ca-deficient solutions depolarize the cells and cause an increase of the membrane resistance (Casteels et al. 1977). We have studied the effect of different concentrations of noradrenaline on the tension development under control conditions, and in a Ca-free solution containing 0.1 mm EGTA. It should be pointed out that the tension response of the isolated pulmonary artery to the same concentration of noradrenaline increases during the first two or three consecutive applications of this substance before reaching a stable value. This phenomenon could be due to the increase of the Ca content in a compartment affected by noradrenaline. Noradrenaline concentrations up to 2-5 x 10-7 M cause, in normal Krebs solution, a mechanical response which creeps up slowly and resembles the slow component depending on external Ca (Bohr, 1963; van Breemen et al. 1973). In Ca-free medium, no response is observed. This finding suggests that, at low noradrenaline concentrations, the excitationcontraction coupling depends on external calcium. Noradrenaline concentrations above 2-5x 10-7M produce a different pattern of tension development resembling the phasic tension development. Moreover, in Ca-free medium tension is also developed, which is of the phasic type (Fig. 4). We can propose, therefore, that at this higher noradrenaline concentration, Ca is released from intracellular binding sites. This hypothesis on the difference between the action of low and high noradrenaline concentrations has been investigated by studying the 45Ca-exchange under the same experimental conditions. First of all, we have determined the amount of "Ca which enters the cellular compartment during an exposure of 10 min to Krebs solutions
EXCITATION-CONTRACTION COUPLING669 containing 1-5 mM-Ca without noradrenaline, or containing 5 x 10- or 10- M of this mediator. The separation of the cellular Ca from the bulk of the tissue Ca is obtained by using the modified La method. It is found that an exposure of the strips (during 10 min) to a Krebs solution containing 45Ca gives an uptake in the so-called La-blocked fraction of 56 cs-moles/kg wet wt. (S.D. of observations = 3, n = 7). This Ca uptake is Control
NOR
Ca-free EGTA
5x 1 0-8 M
2, 5X10-7 M 5x10-7
M
10-6 M
5x10-6M
NOR Krebs
log
NOR EGTA Krebs
5 min
Fig. 4. The effect of different concentrations of noradrenaline on the isometric tension development in Krebs solution (left) and in Ca-free solution containing 0.1 mM-EGTA (right). The latter experiments were performed after 15 min exposure to the Ca-free medium.
increased by 5 x 1O-8M and 106M noradrenaline to 74 (s.D. of observations = 5, n = 7) and 77,umoles/kg wet wt (S.D. of observations = 8, n = 7), respectively. This uptake cannot explain the fact that the tension development is about 10 times larger for the higher noradranaline concentration than for the lower one, as shown in Fig. 4. This dissimilarity in tension development for a similar Ca influx could result from a difference in release of cellular Ca. Such an effect was revealed by the study of the action of these two concentrations of noradrenaline on the 45Ca efflux. As shown in Fig. 5, noradrenaline (5 x 10-8 M) does not affect the Ca efflux, while the higher noradrenaline concentration (10- M) causes a large
R. CASTEELS AND OTHERS
70
increase. These findings support the hypothesis that low concentrations of noradrenaline act by causing only an influx of Ca, while higher concentrations cause, in addition, a release of cellular calcium.
0 10 1-
008 S C %I-
0
._
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0 0 4-
C) cc
0*04 I-
002 1-
0
0
x
l
l
l
l
l
20
40
60
80
100
Time (min)
Fig. 5. Modification of the rate coefficient (min-') of the "5Ca efflux by 5 x 10-8 noradrenaline (0) and by 10-6 M noradrenaline ( x ). The time of the efflux is represented on the abscissa in min. Noradrenaline was added to the efflux medium at min 50, as indicated by the arrow.
Change of [Ca]o, [Na]o and [K]o and their interaction with the noradrenaline effect Na-deficient solution modifies appreciably the effect of noradrenaline on the tension development and on the membrane potential. The question arises whether this change of contractile activity is due to the modification of the effect of noradrenaline on the membrane potential, or whether it is due to
a more
direct interaction of some monovalent ions with the free
71 EXCITATION-CONTRACTION COUPLING cytoplasmic [Ca2+]. We have investigated, therefore, the effects of various experimental conditions, which cause a modification of the membrane potential, on the tension development induced by noradrenaline.
1-5
o 1-0.
0*5
0-
--
11-8 11-8 2-5 50 2-5 [Ca]0 Fig. 6. Tension development of strips of this main pulmonary artery is given as the relative value of the tension induced by the same noradrenaline concentration in Krebs solution. The experimental results are given as mean values + S.D. of observations (n = 5). The open columns (0I) represent the data obtained with 2-5 x 108 M noradrenftline: the shaded columns (T) those obtained with 10-6 M noradrenaline. The concentrations of K and Ca are indicated for each set of columns.
[K]o
2-9
59 50
An increase of [Ca]o to 5 mm hyperpolarizes the smooth muscle membrane from -56 to -59 mV. One could expect that such an increase of [Ca]o would augment the tension response to noradrenaline because of the increase of the inwardly directed Ca gradient. Fig. 6 shows, however, that the tension induced by 2.5 x 10-8 M and 106 M noradrenaline in this Ca excess Krebs solution remains significantly lower than under control conditions. In order to support the hypothesis that the modification of the
R. CASTEELS AND OTHERS 72 response is due largely to the slight hyperpolarization of the membrane, we have also investigated the effect of the same noradrenaline concentrations in a solution containing 5 mM-Ca and 11-6 mM-K. In this solution the membrane potential amounts to -56 mV, a value similar to the one observed under control conditions. Both concentrations of noradrenaline now produce a response which is larger than the control. Another procedure to induce a slight hyperpolarization of the cells consists of reducing A
* e
101*0
V
k--
0
x
05
~
x 0
25
50 Time (min)
x 75
B 1o
.
r0
0-
X 05
0
0
25
50 Time (min)
75
Fig. 7. Tension development of strips of the main pulmonary artery, induced by application of 2-5 x 10-8 M (A) or 106 M (B) noradrenaline during prolonged exposure to various solutions. The tension is given as a relative value of the tension development induced by the same noradrenaline concentration in Krebs solution (dotted line). The time of exposure is given on the abscissa in min. The following solutions have been used in the experiments: Krebs solution containing 1/9 of the usual [Ca]. and 1/4.5 of [Na]o (@); 1/9 [Ca]. and 1/3 [Na]o ( A); 1/9 [Ca]0 and normal [Na]o (0); normal [Ca]0 and 1/4.5 [Na]0 (x).
~ ~ ~ ~ ~ ~ ISV/cmn
73 EXCITATION-CONTRACTION COUPLING [K]0 to a value of 2-9 mM (Casteels et al. 1977). Also, this procedure causes a reduction of the tension response. Exposure of the tissues to a Krebs solution containing 2-5 mM-Ca and 11-8 mM-K depolarizes the cells to -54 mV. Such a small depolarization augments the contractile response to the different concentrations of noradrenaline. A
Caffeine (5
mM)
1 min
YY _ ^ Md
,,,,,,,. .
. . . . . . . . . . . . . . . . .
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. . . .
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_-50/ .1
I
1I
-10
-20
I
-60
/
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20
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0i 4
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--70
-80
Fig. 8. Effect of 5 mm caffeine on the membrane potential, the electrotonic potential and the current-voltage relationship. In the top record (A) it is shown that caffeine depolarizes the cells and reduces the electrotonic potentials produced by inward and outward current pulses. The lower graph (B) represents the current-voltage relationship for the control condition (@) and for tissues exposed to 10 mm caffeine but repolarized by current injection to -57 mV (A).
Simultaneon change of [Ca]o and [Na]o and its effect on the contraction induced by noradrenatine Ca-deficient solutions cause a depolarization of the membrane and an increase of the membrane resistance. These effects were not modified by simultaneous reduction of [Na]0 (Casteels et al., 1977). Because in cardiac muscle the effect of a reduction of [Ca]o on the tension development can be compensated by a reduction of [Na]o we have performed some experiments along these lines. The first experiments in which 2-5 x 1O8M noradrenaline was used
R. CASTEELS AND OTHERS seemed to indicate that a reduction of external Ca and Na, while maintaining the ratio [Ca]: [Na] constant, was more suitable to maintain the mechanical response than a reduction of these ions with a constant ratio [Ca]: [Na]2, as described by Littgau & Niedergerke (1958) for heart muscle. However, a further study of the effect of noradrenaline in solutions with varying [Ca]o and [Na]o has revealed that changing the con74
Caffeine (10 mM)
A
0
0 010 - o
0
^- 0-005 c 1-
a)
._
._j
a)-
M.-
C. 0)
X
ol
0 )9
0
l
~
B
50 Time
(min)
l
100 Caffeine
150
200
(10 mM)
xx9
X5
\X
Xx
)(x
xx
0O(
X X-.X-x
0
I
0
i.
l
I
20 40 30 Time (min) Fig. 9. The effect of 10 mm caffeine on the 42K and 'Cl efflux from the main pulmonary artery. The rate coefficient (min-) is given on the ordinate as a function of the time of efflux in min. The caffeine was added in both experiments as indicated by the arrow. Graph A represents the 42K efflux; graph B, the "C1 efflux. 10
EXCITATION-CONTRACTION COUPLING 75 centration of both these ions exerts a more complex effect than a competition between external Ca and Na for the same binding sites. The results obtained with the two noradrenaline concentrations are represented in Fig. 7. As in several other experimental conditions, there is a change of the tension response as a function of the time of exposure to the solution. In the experiments with 2-5x 10-8 M noradrenaline, the first stimulation gave, for all solutions, a larger response than under control conditions.
008
0 06 _9-
Caffeine (10 mM)
ty
C
-WE ._
._
0
a, 0 0o
004 _
002 _ h0-
0
I
0
20
40
I
I
I
60
80
100
Time (min) Fig. 10. Effect of 10 mm caffeine on the rate coefficient (min-) of the 45Ca efflux from the main pulmonary artery; 10 mm caffeine was added to the solution at min 50 of the efflux as indicated by the arrow.
After 25 min exposure to the respective solutions, the tension development has decreased appreciably below the control value. This decrease is more pronounced for solutions with reduced [Na]0 than with reduced [Ca]o. A simultaneous reduction of [Na]o and [Ca]0 causes a smaller tension decrease. For 10o M noradrenaline, the reduction of [Na]o and [Ca]o act additively to reduce the tension response, but again a reduction of [Na]o causes a
R. CASTEELS AND OTHERS 76 larger depression of the tension than a reduction of [Ca]o. The lowest tension development is observed in solutions with reduced [Ca]o and [Na]o.
Effect of caffeine on the membrane potential and tension response Caffeine, which releases Ca from the sarcoplasmic reticulum of striated muscle, also exerts an important effect on the membrane properties of smooth muscle cells and on their contractile activity. At a concentration Krebs
NOR
2.5x10-7 M
Krebs+10 mm caffeine
j
__ iO3 -
5x10-7 M
2.5x10-6 M 5 min Fig. 11. Tension development of strips of the main pulmonary artery, induced by different concentrations of noradrenaline in normal Krebs solution or in Krebs solution containing 10 mri caffeine.
of 5-10 mM, caffeine depolarizes the cells by 8-10 mV and causes a marked reduction of the amplitude of the electrotonic potential, suggesting a decrease of the membrane resistance (Fig. 8a). A study of the currentvoltage relationships (Fig. 8b) again indicates that the membrane resistance is reduced by the presence of caffeine. The study of the effect of caffeine on the efflux of K and Cl (Fig. 9) reveals that this substance causes an appreciable increase of the rate coefficient for K and Cl efflux. The changes of the membrane potential and of the K and Cl fluxes, are similar to those observed in the presence of 106 M noradrenaline (Casteels et al. 1977). This similarity also applies to the stimulation of the 45Ca-efflux by noradrenaline and caffeine (Fig. 10). However, in contrast with noradrenaline, caffeine does not induce a contraction of the pulmonary artery, and even decreases appreciably the tension development by noradrenaline (Fig. 11).
EXCITATION-CONTRACTION COUPLING
77
DISCUSSION
Our experiments confirm the previous observations made by Su et al. (1964) that, in the main pulmonary artery, a mechanical response can be elicited without depolarization of the membrane. However, we have shown that this type of excitation-contraction coupling occurs only at low concentrations of noradrenaline and that noradrenaline concentrations exceeding 10-7 M also depolarize the cells in a dose-dependent way. We have also studied the sources of activator Ca during stimulation by low and high noradrenaline concentrations. By studying the dependence on external Ca and the tension development induced by noradrenaline concentrations between 5 X 10-8 M and 5 x 106 M noradrenaline, it was found that low noradrenaline concentrations do not elicit a contraction in a Ca-free medium, and that the rising phase of the tension increase in a Ca-containing solution is rather low. Both observations suggest that these low concentrations of noradrenaline act by increasing the Ca-influx. In contrast, high concentrations of noradrenaline also induce a contractile response in Ca-free medium, and the tension development which they produce presents a much faster rising phase, consistent with a partial dependence of this contraction on cellular Ca. Our study of the 45Ca uptake and release during exposure to 5 x 10- M or 106 M noradrenaline has revealed that both concentrations cause a similar increased entry of 45Ca, but that only the higher noradrenaline concentration induces a release of cellular Ca. These findings suggest that, under physiological conditions which are characterized by a low concentration of noradrenaline (Bevan & Su, 1973), the excitation-contraction coupling in the main pulmonary artery depends on the entry of external Ca. The release of cellular Ca seems to be a phenomenon which occurs during exposure to pharmacological concentrations of noradrenaline. The study of the effect of noradrenaline in Na-deficient solution suggested that Na might play a direct role in the excitation-contraction coupling. However, the finding that noradrenaline (instead of depolarizing the cells) induces a slight hyperpolarization made us think of an effect of changes of the membrane potential on the excitation-contraction coupling. We have studied, therefore, the effect of noradrenaline under some experimental conditions which cause a small change of the membrane potential. An increase of [Ca]. to 5 mm hyperpolarizes the cells by about 5 mV and, although at this [Ca]o the inwardly directed Ca gradient is increased, it is observed that the tension induced by any concentration of noradrenaline is lower than in solutions with the normal Ca concentration. The observation that a return to the normal membrane potential, by doubling [K]0, augments the tension response in a solution containing
R. CASTEELS AND OTHERS 78 excess Ca, indicates that the decreased tension development observed in a solution with excess Ca is caused by the hyperpolarization of the membrane. These findings also explain the observations of Hiraoka, Yamagishi & Sano (1968) on the rabbit ear artery, showing that an increase of [Ca]o above 2 mm causes a decrease of the noradrenaline induced contraction. Also, a small hyperpolarization of the membrane, by decreasing [K]O to 2-9 mm, reduces the tension development, while a small depolarization, by doubling [K]O at the usual [Ca]o, augments the tension development. These findings indicate clearly that a small change of the resting potential modifies the tension response to noradrenaline. The effects of a simultaneous reduction of [Ca]o and [Na]o on the tension development are more difficult to interpret. First of all, there occurs a time-dependent decrease of the tension, and secondly, for both noradrenaline concentrations a reduction of [Na]o causes a larger reduction of tension than a reduction of [Ca]o. It is not possible to explain all these changes by assuming a competition between external Ca and Na for the same binding sites. For the experiments with 5 x 108 M noradrenaline, a concomitant reduction of [Ca]o and [Na]o reduces the tension development to a smaller extent than a reduction of [Ca]o or [Na]o only. This could be due to some competition between both ions at the outermembrane. The reduction of the tension development by 5 x 0-8 M noradrenaline in solutions with lowered [Ca]0 could be due to the dependence of the contraction on external Ca. The reduction occurring in Na-deficient solutions is caused probably by hyperpolarization of the membrane, occurring after 5 min exposure to Na-deficient solutions prepared with sucrose. This hyperpolarization is increased slightly by the presence of high concentrations of noradrenaline. The finding that the initial tension development induced by 5 x 10-8 M noradrenaline is larger than the control in all modified solutions could be due to the depolarization of the membrane which occurs in Ca-deficient solutions, and also transiently in Na-deficient solutions prepared with sucrose (Casteels et al., 1977). For experiments with 106 M noradrenaline, the main characteristic is that a reduction of external Na and Ca act additively in reducing the tension. This finding can be explained by the noradrenaline-induced increase of the hyperpolarization in Na-deficient solution, and by the partial dependence of the tension development on external Ca. We can, conclude therefore, that the changes of the external ion composition exert a complex effect on the tension development of the smooth muscle cells of the pulmonary artery consisting of transient or steady changes of the membrane potential, of the availability of external Ca, or of cellular Ca, and in some conditions of a competition between external Ca and Na. The effect of caffeine on the smooth muscle cells of the main pulmonary
EXCITATION-CONTRACTION COUPLING 79 artery cannot be explained at the moment. This substance depolarizes the cells, modifies the ion permeabilities, and releases cellular Ca in much the same way as noradrenaline. However, its contractile effect in normal solutions is very small and it relaxes the tissue during noradrenaline stimulation. It is tempting to relate this action of caffeine to its effect on the sarcoplasmic reticulum of striated muscle (Endo, 1975). However, such a comparison would require a knowledge of the effect of caffeine on the Ca transport of the cell membrane, and of the endoplasmic reticulum in these vascular smooth muscle cells. Such a knowledge is completely lacking at the moment and it is, not possible, therefore, to explain the similarity and dissimilarity between noradrenaline and caffeine. We have, however, to conclude that these findings suggest that still other mechanisms can play a role in the regulation of the excitation-contraction coupling. This work was supported by research grant no. 20.487 of the F.W.G.O. (Belgium) and by a grant of the ministry of Education of Japan (No. 148094), and the Heart Foundation of Japan. REFERENCES BEVAN, J. A. & Su, C. (1973). Sympathetic mechanisms in blood vessels; Nerve and muscle relationships. A. Rev. Pharmac. 13, 269-285. BoHR, D. G. (1963). Vascular smooth muscle: Dual effect of calcium. Science, N.Y. 139, 597-599. CAsmEELs, R., KITAMURA, K., KURIYAMA, H. & SUZUKI, H. (1977). The membrane properties of the smooth muscle cells of the rabbit main pulmonary artery. J. Physiol. 271, 41-61. ENDO, M. (1975). Mechanisms of action of caffeine on the sarcoplasmic reticulum of skeletal muscle. Proc. Japan. Acad. 51, 479-484. HIRAOKA, M., YAMAGISHI, S. & SANo, T. (1968). Role of calcium ions in the contraction of vascular smooth muscle. Am. J. Physiol. 214, 1084-1089. KURIYAMA, H. & TOMITA, T. (1970). The action potential in the smooth muscle of the guinea-pig taenia coli and ureter studied by the double sucrose-gap method. J. gen. Physiol. 55, 147-162. LUTTGAU, H. C. & NIEDERGERKE, R. (1958). The antagonism between Ca and Na on the frog's heart. J. Phy8iol. 143, 486-505. Sommyo, A. V. & SOMLYO, A. P. (1968)- Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Pharmac. exp. Ther. 159, 129-145. Su, C., BEvAN, J. A. & URSILLO, R. C. (1964). Electrical quiescence of pulmonary artery smooth muscle during sympathomimetic stimulation. Circulation Res. 15, 20-27. vAN BREEMEN, C., FARINAS, B. R., CASTEELS, R., GERBA, P., WUYTACK, F. & DEfT, R. (1973). Factors controlling cytoplasmic Ca2+ concentration. Phil. Trans. R. Soc. B, 265, 57-71.
63
EXCITATION-CONTRACTION COUPLING IN THE SMOOTH MUSCLE CELLS OF THE RABBIT MAIN PULMONARY ARTERY
BY R. CASTEELS, K. KITAMURA,* H. KURIYAMA* AND H. SUZUKI* From the Laboratorium voor Fysiologie Universiteit Leuven, B-3000 Leuven, Belgium (Received 6 December 1976) SUMMARY
1. Increasing the external K concentration depolarizes the smooth muscle cells of the main pulmonary artery, and this depolarization reaches a maximal slope of 58 mV for a tenfold change of [K].. The threshold depolarization for inducing contraction is at 4 mV and the maximal contraction is reached at a [K]o of 58 mM. 2. Noradrenaline concentrations between 2 x 10-8 M and 10-7 M induce tension without depolarizing the cells, but at higher concentrations noradrenaline not only elicits a large tension response but also depolarizes the cells in a dose-dependent way. 3. The effect of noradrenaline on the pulmonary artery is appreciably modified by substituting sucrose for NaCl: the cells are slightly hyperpolarized and the tension response is very much reduced. 4. By studying the tension response to noradrenaline in other experimental conditions which cause a small hyperpolarization of the cells, such as 5 mM-[Ca]o, 2.9 mM-[K]o or a small depolarization, such as 11-9 mM-[K]0, it was found that a slight modification of the membrane potential can exert an important effect on the noradrenaline response. 5. A simultaneous decrease of [Ca]0 and [Na]o reduces the tension response to all noradrenaline concentrations. It was found that a reduction of [Na]o exerts a more depressing effect than a reduction of [Ca]o. In interpreting these results we have to take into account changes of the membrane potential, of availability of Ca, and some competition between external Ca and Na. 6. A study of the effect of different concentrations of noradrenaline in Krebs solutions and Ca-free solution has shown that concentrations up to *
Present address: Department of Physiology, Faculty of Dentistry,
Kyushu
University, Fukuoka 812, Japan.
3
P HY 27I
R. CA STEELS AND OTHERS 2*5 x 10-7 M elicit contraction by increasing the Ca influx, while higher concentrations also induce a release of cellular Ca. 7. Caffeine depolarizes the cells and reduces the membrane resistance. It modifies the K, Cl and Ca fluxes in the same way as noradrenaline, but it suppresses the mechanical response induced by noradrenaline. 64
INTRODUCTION
Su, Bevan & Ursillo (1964) have described that noradrenaline induces a contraction in the smooth muscle cells of the main pulmonary artery without depolarizing the cells or eliciting action potentials. In a later study of the effect of noradrenaline on the membrane potential of these cells, Somlyo & Somlyo (1968) have observed (with the sucrose-gap method) that this substance depolarizes and induces oscillations of the membrane. However, because there was only a limited quantitative correlation between the tension development and the depolarization, and because drug-induced contractions could also occur in depolarized tissues, these authors have called this type of excitation-contraction coupling, pharmacomechanical coupling. In addition, the smooth muscle cells of the pulmonary artery can be activated also by depolarizing the cells with K-rich solutions. Ill the present work we have investigated the effect of different concentrations of noradrenaline and of external K on the tension development and on the membrane potential. We have tried to elucidate the role of changes of the membrane potential in the activation of the contractile proteins and to determine the relative importance of external and cellular Ca in eliciting the mechanical response under various experimental conditions. METHODS
The dissections of the strips of the main pulmonary artery and the measurement of the electrical properties of single cells and of K, Cl and Na fluxes have been described in the preceding paper (Casteels, Kitamura, Kuriyama & Suzuki, 1977). The Ca efflux experiments were performed in Krebs solution containing 1*5 mmCa2+, after a loading period in the radioactive solution of 2 hr. The uptake of 4"Ca by the cellular compartment was estimated by using a modification of the La method described by van Breemen, Farinas, Casteels, Gerba, Wuytack & Deth (1973). After the loading procedure, the tissues were first washed for 5 min in an isotonic Ca-free solution containing 10 m -La. The further efflux was then performed in the same solution, but added with 0.1 mm DNP and 1 mm iodo-acetic acid. This procedure induces a slowing down of the efflux of 4"Ca from the cellular compartment and therefore facilitates the estimation of the amount of 4"Ca in the cellular compartment by extrapolating the slow phase of the efflux to zero time. In order to record simultaneously the electrical and the mechanical activity, we have used the double sucrose-gap method as described by Kuriyama & Tomita (1970). The tension development of the tissues was measured under isometric conditions
EXCITATION-CONTRACTION COUPLING
65 in an organ bath of 3 ml. which was perfused continuously at a rate of 6 ml./min. The temperature was maintained at 35 'C. Both ends of the preparation were tied by cotton strings between a tension transducer and a hook at the bottom of the bath. The composition of the physiological solution, and of the modified solutions, have been described (Casteels et al. 1977). The drugs used in the experiments were noradrenaline HCl, caffeine, EGTA (ethylene glycol bis (fl-aminoethyl ether)-N-N'tetraacetic acid) and ouabain (g-strophantin). The concentrations used in the experiments are given in the Results. RESULTS
Contraction induced by change of [K]o Action potentials normally, are not generated in smooth muscle cells of the pulmonary artery and the mechanical response is induced in these cells either by a depolarization of the membrane or by pharmacomechanical (o)
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100
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Fig. 1. The changes of the membrane potential measured with intracellular electrodes in mV (0) and of the mechanical response (x) induced by different concentrations [K]0, are represented as a function of the log of [K]O. The tension development of the different [K]o is given as a relative value of the tension developed in 137 mM-[K]0.
coupling, which can occur with or without depolarization. In order to investigate the effect of the membrane potential on the mechanical response, we have studied the action of various concentrations [K]0 on the 3-2
66 R. CJASTEELS AND OTHERS membrane potential and tension development. Theseresults arerepresented in Fig. 1. The minimal depolarization to evoke contraction by increasing [K]0 is 4 mV and such a depolarization is obtained at a [K]o of 9 mm. A further increase of [K]o enlarges the amplitude of the tension development, reaching its max. at 58 mm. The graph representing the membrane potential as a function of the logarithm of [K]o has a maximal slope of 58 mV per tenfold change of [K]o. The cells slightly hyperpolarize by
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-15
Fig. 2. Relationship between the membrane potential (mV) and the mechanical response (mg) measured with the double sucrose-gap method. The membrane potential was displaced by application of outward current of various intensities. The membrane potential of three different preparations is represented in the graph by different symbols (0O 0, x ). The upper traces show the actual potential changes and the mechanical responses produced by three different intensities of the outward current pulses.
reducing [K]o to 3 mm, while a further reduction of [K]o results again in a depolarization by some mV and produces a small tension development. Using the double sucrose-gap method we have also investigated the relationship between depolarization of the membrane induced by current injection and the mechanical response (Fig. 2). The membrane potential measured by this method in three different preparations was - 48, -47
EXCITATION-CONTRACTION COUPLING 67 and -42 mV. The lower value of these measurements as compared with the ones obtained with micro-electrodes is due presumably to leakage of current. The depolarization which had to be induced in these preparations to produce contraction were 4, 6 and 3 mV, respectively. We can assume, therefore, that the critical depolarization for inducing contraction is about 4 mV, as suggested already by the experiments with excess [K]o.
-45
2
-50 T
E
Na-defic entA
~0
2.5 x10-6 2-5x10-7 5X10-6 5x10-7 Noradrenaline (M) Fig. 3. Effects of various concentrations of noradrenaline between 2-5 x 10-8 and 5 x 10-6 M on the membrane potential and tension development in Krebs solution and in Na-deficient solution prepared with sucrose. The membrane potential (mV) measured with intracellular electrodes, is given as mean values + S.D. of observatoins of 11-18 penetrations in Krebs solution (0) and in Na-deficient solution (e). The mechanical response is represented by A for the values obtained in Krebs solution and by A for values in Na-deficient solution. Each point represents the mean of seven experiments. 2.5x10-8 Control 5x10-8 Na-deficient
Contraction induced by noradrenaline in Krebs solution We have investigated the effect of different concentrations of noradrenaline between 5 x 10-9 and 5 x 106 M on the membrane potential and tension development of the pulmonary artery. Concentrations of noradrenaline between 2 x 108 M and 10-7 M induce a tension development of the pulmonary artery without affecting the membrane potential. Higher concentrations of noradrenaline cause a progressively increasing tension development, but also depolarize the cells as represented in Fig. 3.
R. CASTEELS AND OTHERS 68 At 5 x 106 M this depolarization reaches a value of 9 mV. This depolarizing action of high concentrations of noradrenaline is due probably to an increase of the permeability of the membrane for Na and Cl, while the concomitant reduction of the membrane resistance depends on the increase of the permeability of the membrane for Na, C1 andK (Casteels et al. 1977). It was found worthwhile therefore, to study the effect of different concentrations of noradrenaline in Na- and Cl-deficient medium, prepared by substituting sucrose for NaCl. This solution only slightly hyperpolarizes the cells, but changes appreciably the effect of noradrenaline on the membrane potential and tension development. An increase of the noradrenaline concentration slightly augments the hyperpolarization of the cells and causes a tension development which remains much smaller over the whole range of the noradrenaline concentration. In addition, the threshold concentration for inducing contraction is shifted up to 2-5 x 10-7 M-
noradrenaline.
Interaction of [Ca]o and noradrenaline in inducing contraction Ca-deficient solutions depolarize the cells and cause an increase of the membrane resistance (Casteels et al. 1977). We have studied the effect of different concentrations of noradrenaline on the tension development under control conditions, and in a Ca-free solution containing 0.1 mm EGTA. It should be pointed out that the tension response of the isolated pulmonary artery to the same concentration of noradrenaline increases during the first two or three consecutive applications of this substance before reaching a stable value. This phenomenon could be due to the increase of the Ca content in a compartment affected by noradrenaline. Noradrenaline concentrations up to 2-5 x 10-7 M cause, in normal Krebs solution, a mechanical response which creeps up slowly and resembles the slow component depending on external Ca (Bohr, 1963; van Breemen et al. 1973). In Ca-free medium, no response is observed. This finding suggests that, at low noradrenaline concentrations, the excitationcontraction coupling depends on external calcium. Noradrenaline concentrations above 2-5x 10-7M produce a different pattern of tension development resembling the phasic tension development. Moreover, in Ca-free medium tension is also developed, which is of the phasic type (Fig. 4). We can propose, therefore, that at this higher noradrenaline concentration, Ca is released from intracellular binding sites. This hypothesis on the difference between the action of low and high noradrenaline concentrations has been investigated by studying the 45Ca-exchange under the same experimental conditions. First of all, we have determined the amount of "Ca which enters the cellular compartment during an exposure of 10 min to Krebs solutions
EXCITATION-CONTRACTION COUPLING669 containing 1-5 mM-Ca without noradrenaline, or containing 5 x 10- or 10- M of this mediator. The separation of the cellular Ca from the bulk of the tissue Ca is obtained by using the modified La method. It is found that an exposure of the strips (during 10 min) to a Krebs solution containing 45Ca gives an uptake in the so-called La-blocked fraction of 56 cs-moles/kg wet wt. (S.D. of observations = 3, n = 7). This Ca uptake is Control
NOR
Ca-free EGTA
5x 1 0-8 M
2, 5X10-7 M 5x10-7
M
10-6 M
5x10-6M
NOR Krebs
log
NOR EGTA Krebs
5 min
Fig. 4. The effect of different concentrations of noradrenaline on the isometric tension development in Krebs solution (left) and in Ca-free solution containing 0.1 mM-EGTA (right). The latter experiments were performed after 15 min exposure to the Ca-free medium.
increased by 5 x 1O-8M and 106M noradrenaline to 74 (s.D. of observations = 5, n = 7) and 77,umoles/kg wet wt (S.D. of observations = 8, n = 7), respectively. This uptake cannot explain the fact that the tension development is about 10 times larger for the higher noradranaline concentration than for the lower one, as shown in Fig. 4. This dissimilarity in tension development for a similar Ca influx could result from a difference in release of cellular Ca. Such an effect was revealed by the study of the action of these two concentrations of noradrenaline on the 45Ca efflux. As shown in Fig. 5, noradrenaline (5 x 10-8 M) does not affect the Ca efflux, while the higher noradrenaline concentration (10- M) causes a large
R. CASTEELS AND OTHERS
70
increase. These findings support the hypothesis that low concentrations of noradrenaline act by causing only an influx of Ca, while higher concentrations cause, in addition, a release of cellular calcium.
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l
l
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l
l
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40
60
80
100
Time (min)
Fig. 5. Modification of the rate coefficient (min-') of the "5Ca efflux by 5 x 10-8 noradrenaline (0) and by 10-6 M noradrenaline ( x ). The time of the efflux is represented on the abscissa in min. Noradrenaline was added to the efflux medium at min 50, as indicated by the arrow.
Change of [Ca]o, [Na]o and [K]o and their interaction with the noradrenaline effect Na-deficient solution modifies appreciably the effect of noradrenaline on the tension development and on the membrane potential. The question arises whether this change of contractile activity is due to the modification of the effect of noradrenaline on the membrane potential, or whether it is due to
a more
direct interaction of some monovalent ions with the free
71 EXCITATION-CONTRACTION COUPLING cytoplasmic [Ca2+]. We have investigated, therefore, the effects of various experimental conditions, which cause a modification of the membrane potential, on the tension development induced by noradrenaline.
1-5
o 1-0.
0*5
0-
--
11-8 11-8 2-5 50 2-5 [Ca]0 Fig. 6. Tension development of strips of this main pulmonary artery is given as the relative value of the tension induced by the same noradrenaline concentration in Krebs solution. The experimental results are given as mean values + S.D. of observations (n = 5). The open columns (0I) represent the data obtained with 2-5 x 108 M noradrenftline: the shaded columns (T) those obtained with 10-6 M noradrenaline. The concentrations of K and Ca are indicated for each set of columns.
[K]o
2-9
59 50
An increase of [Ca]o to 5 mm hyperpolarizes the smooth muscle membrane from -56 to -59 mV. One could expect that such an increase of [Ca]o would augment the tension response to noradrenaline because of the increase of the inwardly directed Ca gradient. Fig. 6 shows, however, that the tension induced by 2.5 x 10-8 M and 106 M noradrenaline in this Ca excess Krebs solution remains significantly lower than under control conditions. In order to support the hypothesis that the modification of the
R. CASTEELS AND OTHERS 72 response is due largely to the slight hyperpolarization of the membrane, we have also investigated the effect of the same noradrenaline concentrations in a solution containing 5 mM-Ca and 11-6 mM-K. In this solution the membrane potential amounts to -56 mV, a value similar to the one observed under control conditions. Both concentrations of noradrenaline now produce a response which is larger than the control. Another procedure to induce a slight hyperpolarization of the cells consists of reducing A
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x 75
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Fig. 7. Tension development of strips of the main pulmonary artery, induced by application of 2-5 x 10-8 M (A) or 106 M (B) noradrenaline during prolonged exposure to various solutions. The tension is given as a relative value of the tension development induced by the same noradrenaline concentration in Krebs solution (dotted line). The time of exposure is given on the abscissa in min. The following solutions have been used in the experiments: Krebs solution containing 1/9 of the usual [Ca]. and 1/4.5 of [Na]o (@); 1/9 [Ca]. and 1/3 [Na]o ( A); 1/9 [Ca]0 and normal [Na]o (0); normal [Ca]0 and 1/4.5 [Na]0 (x).
~ ~ ~ ~ ~ ~ ISV/cmn
73 EXCITATION-CONTRACTION COUPLING [K]0 to a value of 2-9 mM (Casteels et al. 1977). Also, this procedure causes a reduction of the tension response. Exposure of the tissues to a Krebs solution containing 2-5 mM-Ca and 11-8 mM-K depolarizes the cells to -54 mV. Such a small depolarization augments the contractile response to the different concentrations of noradrenaline. A
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Fig. 8. Effect of 5 mm caffeine on the membrane potential, the electrotonic potential and the current-voltage relationship. In the top record (A) it is shown that caffeine depolarizes the cells and reduces the electrotonic potentials produced by inward and outward current pulses. The lower graph (B) represents the current-voltage relationship for the control condition (@) and for tissues exposed to 10 mm caffeine but repolarized by current injection to -57 mV (A).
Simultaneon change of [Ca]o and [Na]o and its effect on the contraction induced by noradrenatine Ca-deficient solutions cause a depolarization of the membrane and an increase of the membrane resistance. These effects were not modified by simultaneous reduction of [Na]0 (Casteels et al., 1977). Because in cardiac muscle the effect of a reduction of [Ca]o on the tension development can be compensated by a reduction of [Na]o we have performed some experiments along these lines. The first experiments in which 2-5 x 1O8M noradrenaline was used
R. CASTEELS AND OTHERS seemed to indicate that a reduction of external Ca and Na, while maintaining the ratio [Ca]: [Na] constant, was more suitable to maintain the mechanical response than a reduction of these ions with a constant ratio [Ca]: [Na]2, as described by Littgau & Niedergerke (1958) for heart muscle. However, a further study of the effect of noradrenaline in solutions with varying [Ca]o and [Na]o has revealed that changing the con74
Caffeine (10 mM)
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20 40 30 Time (min) Fig. 9. The effect of 10 mm caffeine on the 42K and 'Cl efflux from the main pulmonary artery. The rate coefficient (min-) is given on the ordinate as a function of the time of efflux in min. The caffeine was added in both experiments as indicated by the arrow. Graph A represents the 42K efflux; graph B, the "C1 efflux. 10
EXCITATION-CONTRACTION COUPLING 75 centration of both these ions exerts a more complex effect than a competition between external Ca and Na for the same binding sites. The results obtained with the two noradrenaline concentrations are represented in Fig. 7. As in several other experimental conditions, there is a change of the tension response as a function of the time of exposure to the solution. In the experiments with 2-5x 10-8 M noradrenaline, the first stimulation gave, for all solutions, a larger response than under control conditions.
008
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I
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Time (min) Fig. 10. Effect of 10 mm caffeine on the rate coefficient (min-) of the 45Ca efflux from the main pulmonary artery; 10 mm caffeine was added to the solution at min 50 of the efflux as indicated by the arrow.
After 25 min exposure to the respective solutions, the tension development has decreased appreciably below the control value. This decrease is more pronounced for solutions with reduced [Na]0 than with reduced [Ca]o. A simultaneous reduction of [Na]o and [Ca]0 causes a smaller tension decrease. For 10o M noradrenaline, the reduction of [Na]o and [Ca]o act additively to reduce the tension response, but again a reduction of [Na]o causes a
R. CASTEELS AND OTHERS 76 larger depression of the tension than a reduction of [Ca]o. The lowest tension development is observed in solutions with reduced [Ca]o and [Na]o.
Effect of caffeine on the membrane potential and tension response Caffeine, which releases Ca from the sarcoplasmic reticulum of striated muscle, also exerts an important effect on the membrane properties of smooth muscle cells and on their contractile activity. At a concentration Krebs
NOR
2.5x10-7 M
Krebs+10 mm caffeine
j
__ iO3 -
5x10-7 M
2.5x10-6 M 5 min Fig. 11. Tension development of strips of the main pulmonary artery, induced by different concentrations of noradrenaline in normal Krebs solution or in Krebs solution containing 10 mri caffeine.
of 5-10 mM, caffeine depolarizes the cells by 8-10 mV and causes a marked reduction of the amplitude of the electrotonic potential, suggesting a decrease of the membrane resistance (Fig. 8a). A study of the currentvoltage relationships (Fig. 8b) again indicates that the membrane resistance is reduced by the presence of caffeine. The study of the effect of caffeine on the efflux of K and Cl (Fig. 9) reveals that this substance causes an appreciable increase of the rate coefficient for K and Cl efflux. The changes of the membrane potential and of the K and Cl fluxes, are similar to those observed in the presence of 106 M noradrenaline (Casteels et al. 1977). This similarity also applies to the stimulation of the 45Ca-efflux by noradrenaline and caffeine (Fig. 10). However, in contrast with noradrenaline, caffeine does not induce a contraction of the pulmonary artery, and even decreases appreciably the tension development by noradrenaline (Fig. 11).
EXCITATION-CONTRACTION COUPLING
77
DISCUSSION
Our experiments confirm the previous observations made by Su et al. (1964) that, in the main pulmonary artery, a mechanical response can be elicited without depolarization of the membrane. However, we have shown that this type of excitation-contraction coupling occurs only at low concentrations of noradrenaline and that noradrenaline concentrations exceeding 10-7 M also depolarize the cells in a dose-dependent way. We have also studied the sources of activator Ca during stimulation by low and high noradrenaline concentrations. By studying the dependence on external Ca and the tension development induced by noradrenaline concentrations between 5 X 10-8 M and 5 x 106 M noradrenaline, it was found that low noradrenaline concentrations do not elicit a contraction in a Ca-free medium, and that the rising phase of the tension increase in a Ca-containing solution is rather low. Both observations suggest that these low concentrations of noradrenaline act by increasing the Ca-influx. In contrast, high concentrations of noradrenaline also induce a contractile response in Ca-free medium, and the tension development which they produce presents a much faster rising phase, consistent with a partial dependence of this contraction on cellular Ca. Our study of the 45Ca uptake and release during exposure to 5 x 10- M or 106 M noradrenaline has revealed that both concentrations cause a similar increased entry of 45Ca, but that only the higher noradrenaline concentration induces a release of cellular Ca. These findings suggest that, under physiological conditions which are characterized by a low concentration of noradrenaline (Bevan & Su, 1973), the excitation-contraction coupling in the main pulmonary artery depends on the entry of external Ca. The release of cellular Ca seems to be a phenomenon which occurs during exposure to pharmacological concentrations of noradrenaline. The study of the effect of noradrenaline in Na-deficient solution suggested that Na might play a direct role in the excitation-contraction coupling. However, the finding that noradrenaline (instead of depolarizing the cells) induces a slight hyperpolarization made us think of an effect of changes of the membrane potential on the excitation-contraction coupling. We have studied, therefore, the effect of noradrenaline under some experimental conditions which cause a small change of the membrane potential. An increase of [Ca]. to 5 mm hyperpolarizes the cells by about 5 mV and, although at this [Ca]o the inwardly directed Ca gradient is increased, it is observed that the tension induced by any concentration of noradrenaline is lower than in solutions with the normal Ca concentration. The observation that a return to the normal membrane potential, by doubling [K]0, augments the tension response in a solution containing
R. CASTEELS AND OTHERS 78 excess Ca, indicates that the decreased tension development observed in a solution with excess Ca is caused by the hyperpolarization of the membrane. These findings also explain the observations of Hiraoka, Yamagishi & Sano (1968) on the rabbit ear artery, showing that an increase of [Ca]o above 2 mm causes a decrease of the noradrenaline induced contraction. Also, a small hyperpolarization of the membrane, by decreasing [K]O to 2-9 mm, reduces the tension development, while a small depolarization, by doubling [K]O at the usual [Ca]o, augments the tension development. These findings indicate clearly that a small change of the resting potential modifies the tension response to noradrenaline. The effects of a simultaneous reduction of [Ca]o and [Na]o on the tension development are more difficult to interpret. First of all, there occurs a time-dependent decrease of the tension, and secondly, for both noradrenaline concentrations a reduction of [Na]o causes a larger reduction of tension than a reduction of [Ca]o. It is not possible to explain all these changes by assuming a competition between external Ca and Na for the same binding sites. For the experiments with 5 x 108 M noradrenaline, a concomitant reduction of [Ca]o and [Na]o reduces the tension development to a smaller extent than a reduction of [Ca]o or [Na]o only. This could be due to some competition between both ions at the outermembrane. The reduction of the tension development by 5 x 0-8 M noradrenaline in solutions with lowered [Ca]0 could be due to the dependence of the contraction on external Ca. The reduction occurring in Na-deficient solutions is caused probably by hyperpolarization of the membrane, occurring after 5 min exposure to Na-deficient solutions prepared with sucrose. This hyperpolarization is increased slightly by the presence of high concentrations of noradrenaline. The finding that the initial tension development induced by 5 x 10-8 M noradrenaline is larger than the control in all modified solutions could be due to the depolarization of the membrane which occurs in Ca-deficient solutions, and also transiently in Na-deficient solutions prepared with sucrose (Casteels et al., 1977). For experiments with 106 M noradrenaline, the main characteristic is that a reduction of external Na and Ca act additively in reducing the tension. This finding can be explained by the noradrenaline-induced increase of the hyperpolarization in Na-deficient solution, and by the partial dependence of the tension development on external Ca. We can, conclude therefore, that the changes of the external ion composition exert a complex effect on the tension development of the smooth muscle cells of the pulmonary artery consisting of transient or steady changes of the membrane potential, of the availability of external Ca, or of cellular Ca, and in some conditions of a competition between external Ca and Na. The effect of caffeine on the smooth muscle cells of the main pulmonary
EXCITATION-CONTRACTION COUPLING 79 artery cannot be explained at the moment. This substance depolarizes the cells, modifies the ion permeabilities, and releases cellular Ca in much the same way as noradrenaline. However, its contractile effect in normal solutions is very small and it relaxes the tissue during noradrenaline stimulation. It is tempting to relate this action of caffeine to its effect on the sarcoplasmic reticulum of striated muscle (Endo, 1975). However, such a comparison would require a knowledge of the effect of caffeine on the Ca transport of the cell membrane, and of the endoplasmic reticulum in these vascular smooth muscle cells. Such a knowledge is completely lacking at the moment and it is, not possible, therefore, to explain the similarity and dissimilarity between noradrenaline and caffeine. We have, however, to conclude that these findings suggest that still other mechanisms can play a role in the regulation of the excitation-contraction coupling. This work was supported by research grant no. 20.487 of the F.W.G.O. (Belgium) and by a grant of the ministry of Education of Japan (No. 148094), and the Heart Foundation of Japan. REFERENCES BEVAN, J. A. & Su, C. (1973). Sympathetic mechanisms in blood vessels; Nerve and muscle relationships. A. Rev. Pharmac. 13, 269-285. BoHR, D. G. (1963). Vascular smooth muscle: Dual effect of calcium. Science, N.Y. 139, 597-599. CAsmEELs, R., KITAMURA, K., KURIYAMA, H. & SUZUKI, H. (1977). The membrane properties of the smooth muscle cells of the rabbit main pulmonary artery. J. Physiol. 271, 41-61. ENDO, M. (1975). Mechanisms of action of caffeine on the sarcoplasmic reticulum of skeletal muscle. Proc. Japan. Acad. 51, 479-484. HIRAOKA, M., YAMAGISHI, S. & SANo, T. (1968). Role of calcium ions in the contraction of vascular smooth muscle. Am. J. Physiol. 214, 1084-1089. KURIYAMA, H. & TOMITA, T. (1970). The action potential in the smooth muscle of the guinea-pig taenia coli and ureter studied by the double sucrose-gap method. J. gen. Physiol. 55, 147-162. LUTTGAU, H. C. & NIEDERGERKE, R. (1958). The antagonism between Ca and Na on the frog's heart. J. Phy8iol. 143, 486-505. Sommyo, A. V. & SOMLYO, A. P. (1968)- Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Pharmac. exp. Ther. 159, 129-145. Su, C., BEvAN, J. A. & URSILLO, R. C. (1964). Electrical quiescence of pulmonary artery smooth muscle during sympathomimetic stimulation. Circulation Res. 15, 20-27. vAN BREEMEN, C., FARINAS, B. R., CASTEELS, R., GERBA, P., WUYTACK, F. & DEfT, R. (1973). Factors controlling cytoplasmic Ca2+ concentration. Phil. Trans. R. Soc. B, 265, 57-71.
