Involvement of Ca2+ influx-induced Ca2+ release in contractions of intact vascular smooth muscles KATSUAKI
ITO,
TAKAAKI
IKEMOTO,
AND
SACHIKO
TAKAKURA
Department of Veterinary Pharmacology, Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan
lease of Ca2+ from SR in vascular smooth muscles (9,10, 13). Unlike procaine (1) or caffeine (18, 22, 32), ryanodine acts selectively on the SR because it does not directly affect either transmembrane Ca2+ influx or the contractile machinery (17, 33). Therefore the sensitivity isotonic KC1 solution induced a contraction (Ca contraction), of a contraction to ryanodine can be interpreted as the a part of which dependedon preloading the intracellular stores with Ca*’ and which was sensitive to 3 x 10m5 M ryanodine. presence of a component associated with Ca2’ release Because‘%a*+ influx from the external fluid upon the addition from the SR. We have used ryanodine as a tool to analyze Ca2+ chanof Ca*’ was not modified either by the state of filling of the how Ca2’ influx through voltage-dependent Ca*’ store or by the presence of ryanodine, the ryanodine- nels causes Ca2+ release in intact guinea pig aortic sensitive component of the contraction could be attributed to smooth muscles depolarized by high K+. ITO, KATSUAKI, TAKAAKI IKEMOTO, AND SACHIKO TAKACa*’ release in conKURA. Involvement of Ca*+ influx-induced tractions of intact vascular smooth muscles. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1464-H1470,1991.-Application of Ca*+ (0.1-2.5 mM) to guinea pig aortas incubated in Ca*‘-free
the Ca*+influx-induced Ca*’ releasefrom the Ca*’ store. Supporting the possibility of involvement of Ca*‘-induced Ca*’ release,the Ca contraction due to 0.1 mM Ca*’ was enhanced either by decreasingMg2+in the mediumor by low temperature. The ratio of the ryanodine-sensitive fraction in the Ca contraction was inversely related to the concentration of Ca*’ added and also to the extent of 45Ca2+ influx. When the Ca*’ influx wasdecreasedby verapamil or cadmium,the ratio of ryanodinesensitive fraction increased. On the contrary, an increase of Ca*’ influx by CGP-28392 decreasedthe ratio. These results suggestthat Ca*’ influx at a physiological level triggers Ca*’ releasefrom the Ca*’ store, resulting in the amplification of contractile force.
METHODS
Preparations. Guinea pigs weighing 250-450 g were stunned by a blow on the neck, and the thoracic aortas were isolated. Rectangular strips of aorta were made from aortic rings of 2-3 mm in width. The muscles were suspended in an organ bath containing 5 ml physiological saline solution (PSS; in mM: 136.8 NaCl, 5.4 KCl, 2.5 CaC12, 0.5 MgC12, 11.9 NaHC03, and 5.5 dextrose, pH 7.3-7.4) gassed with 95% 02-5% CO2 (37°C unless otherwise stated). They were equilibrated in PSS for 1 h before experiments, and a basal tension of 1 g was sarcoplasmicreticulum; calcium channel; depolarized muscle; maintained. guineapig aorta; ryanodine Tension experiments. The Ca2’-induced contraction (termed as Ca contraction) was elicited by reintroduction of Ca2+ at various concentrations to muscles preincuIT IS NOW WIDELY ACCEPTED that the Ca2+-induced bated in nominally Ca2’- free isotonic KC1 solution (Orelease of Ca2+ from sarcoplasmic reticulum (SR) can Ca iso-K solution; in mM: 142.2 KCl, 0.5 MgC12, 11.9 occur in every type of muscle (6). This can be demonNaHCOs, and 5.5 dextrose) for 20 min. Before exposure strated experimentally as a caffeine-induced contraction. to 0-Ca iso-K solution, the intracellular Ca2+ store of the Many vascular smooth muscles respond to caffeine with muscle was loaded with Ca2’ to different degrees, i.e., a transient contraction in intact or skinned preparations, normal loading, excess loading, or depletion of Ca2+ from suggesting the existence of a Ca2+-induced Ca2’ release the store. To normally load the store, the muscles were mechanism (14, 15). However, the manifestation of caf- exposed to normal PSS for 15 min after lo-min preinfeine-induced contraction does not necessarily mean that cubation in Ca2+-free PSS. If the store was loaded with the Ca2+-induced Ca2+ release mechanism plays a signifCa2’ in 2.5 mM Ca2+-PSS for more than 5 min, a conicant role under physiological conditions in intact vas- stant Ca contraction was obtained. Unless specified, the cular smooth muscles. To assess whether this mechanism muscles were loaded with Ca2+ in this way. Excess loadis physiologically important in a given muscle, the ability ing was achieved by exposing the aorta to hypertonically of Ca2+ influx across the plasma membrane to cause Ca2’ added 60 mM KC1 in the presence of 2.5 mM Ca2+ for 15 release from the SR must be demonstrated. This is an min until the external medium was switched to O-Ca isoimportant issue because various transmitters and hor- K solution. The effectiveness of this procedure was conmones cause both transmembrane Ca2+ influx and intrafirmed by the experiment that this procedure potentiated cellular Ca2+ release after activation of the receptors in the contraction due to norepinephrine applied in Ca2+vascular smooth muscles. free PSS to 170% of that after normal loading. Ca2’ Ryanodine ultimately abolishes the Ca2+-induced re- deprivation was achieved by consecutive exposures to H1464
0363-6135/91
$1.50
Copyright 0 1991 the American Physiological Society
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cA2+
INFLUX
AND
CA2+
RELEASE
norepinephrine ( 10m6M) or caffeine (30 mM) in Ca2’free PSS until the phasic contraction due to norepinephrine or caffeine was abolished. 45Ca2+uptake experiments. Cellular 45Ca2+ uptake into aortic smooth muscles was measured by the cold lanthanum washing method (19). A thoracic aorta was divided into four strips, each of which was 8-12 mg wet wt. Muscles were loaded with 1 g of tension in a circular direction and equilibrated in normal tris(hydroxymethyl)aminomethane (Tris)-PSS containing (in mM) 136.8 NaCl, 5.4 KCl, 2.5 CaC12, 0.5 MgC12, 5.5 dextrose, and 5.0 Tris-HCl (pH 7.4) and gassed with 100% O2 for 60 min at 37OC. During the equilibration period muscles were conditioned with hypertonic 65.4 mM KC1 for between 15 and 30 min. The muscles were transferred to Ca2+-free isotonic KC1 solution (buffered with 5 mM Tris). Twenty minutes later 0.1, 0.5, 1.0 or 2.5 mM Ca2’ with 45Ca2+ (1 &i/ml) was applied. Five or 30 min later the muscles were transferred to La3+ solution (in mM: 80.8 LaC13, 5.5 dextrose, and 5 Tris, pH 6.8) cooled to below 2°C to displace the extracellular Ca2+ during the 60-min immersion. After that, the muscle was blotted between two filter papers (Whatman no. 2) under a l5g/cm2 weight for 10 s, and the wet weight was measured on a torsion balance. The residual radioactivity was counted as cellular 45Ca2+ uptake. Reagents. The following drugs were used: ryanodine (lot 704RWP-1, S. B. Penick, Lyndhurst, NJ), norepinephrine (Sigma, St. Louis, MO), verapamil (Eisai, Tokyo, Japan), and CGP-28392 (Ciba-Geigy, Basel, Switzerland). CGP-28392 was dissolved in dimethyl sulfoxide; other drugs were dissolved in distilled water. Statistics. Data are expressed as mean t SE. Significance was considered at the level of P < 0.05.
IN VASCULAR
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Ca2+-induced contraction of guinea pig aorta preincubated in Ca2+-free, isotonic KC1 solution. Before addition of Ca2’ intracellular Ca2+ store was overloaded or normally loaded with Ca2’ or depleted of Ca2’ (see METHODS). At 0 min 0.1 mM Ca2’ was added. One hundred percent in the ordinate represents maximum contraction induced by hypertonically added 60 mM KC1 to normal physiological saline solution (PSS). Filled circles, control; open circles, in the presence of 3 X lo-” M ryanodine, which was applied 20 min before addition of Ca2’. Each point represents mean t SE of 6-10 preparations. * Significantly different from control (P < 0.05). FIG.
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Ca2’ to vascular smooth muscle incubated in a Ca2+-free high-K+ solution causes transmembrane Ca2’ influx and induces a contraction. A simplistic view of this event is that Ca2+ coming from the external milieu directly activates the contractile machinery. However, the Ca2’ can also trigger the release of Ca2’ from SR. Our previous papers (12, 13) showed that a fraction of the Ca2+induced contraction of depolarized guinea pig aorta was sensitive to ryanodine, suggesting that Ca2+ release was involved in the contraction. In this study we analyzed the detailed nature of the Ca contraction. Figure 1 shows the time course of 0.1 mM Ca2+-induced contraction over a lo-min period in guinea pig aortas, which had been preincubated in 0-Ca iso-K solution, under three states of Ca2+ loading of the Ca2’ store, i.e., overloading, normal loading, or depletion of Ca2+. As shown in Fig. 1, the contraction in Ca2+-overloaded muscles rose rapidly with a large amplitude upon the addition of 0.1 mM Ca2+. On the other hand, the tension development in Ca2+ -depleted muscles was very slow and small. The rate of rise and the amplitude of the Ca contraction in normally loaded muscles were intermediate. Ryanodine (3 x 10m5 M) inhibited the development
k&ed
2
RESULTS
Dependence of Ca2+-induced contraction of depolarized aorta on Ca2+ loading of Ca2+ store. The application of
H1465
MUSCLE
30 min
-il
l .*.*.v l .*.*.v l .*.v.* v.*.v 5v.v v.*.v l .v.v .*.*.v ll .*.*.v v.*.*.* y normally loaded > Ca2+-depleted muscle. Especially, in Ca2+-depleted muscle, ryanodine did not significantly decrease the Ca contraction during first 5 min. 45Ca2+uptake into Ca2+-loaded muscles and Ca2+-depleted muscles. With regard to the different magnitudes
of responses of depolarized aorta to added Ca2+, it was possible that the degree of Ca2’ influx was different between Ca2+-loaded and Ca2’-depleted muscles, since Putney (27) postulated that Ca2’ influx depended on the emptying of Ca2+ stores in some cells. To test this, 45Ca2+ uptake into Ca2’ -overloaded and Ca2+-depleted muscles was measured. Figure 2 shows the 45Ca2+ uptake into depolarized aortas during a 0.1 mM Ca2+-induced contraction. The 45Ca2+ influx in the absence of ryanodine was not different between Ca2’-loaded and Ca2+-depleted muscles. Furthermore, ryanodine did not affect the 45Ca
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H1466
CA*+
INFLUX
AND
CA*+
RELEASE
influx for 5 or 30 min after the addition of Ca2’. These results suggest that the greater response to Ca2’ of the Ca2+-loaded muscle was not due to augmented Ca2’ influx and also that the greater sensitivity to ryanodine in such muscle was not due to inhibition of Ca2’ influx by ryanodine. Some characteristics of the Ca contraction. The above results suggest that Ca2+- induced Ca2’ release is involved in the contraction induced by the addition of 0.1 mM Ca2’ into 0-Ca iso-K solution. Three further tests were performed to determine whether the Ca contraction due to 0.1 mM Ca2’ had some characteristics of Ca2’-induced Ca2+ release. Mg2+ modulates the Ca2’ -induced Ca2’ release mechanism (6). In fact, contractions of smooth muscles due to caffeine were reportedly potentiated by incubation of muscle in Mg’-deficient solution (18, 30, 32). If the Ca2’-induced Ca2’ release mechanism operates in the Ca contraction elicited by the protocol used in this study, the contraction should be enhanced by incubation with Mg’-free PSS. Figure 3 shows that this is the case. When the muscle was equilibrated in Mg+-free PSS for >l h and then exposed to Ca2’- and Mg+-free isotonic KC1 solution, the contraction induced by 0.1 mM Ca2+ was larger compared with that preincubated with 1 mM Mg+, whereas the contraction induced by 5 mM Ca2’ was the same or slightly depressed in Mg2’-deficient solution compared with Mg’-containing PSS. Thus the contraction of depolarized aorta induced by 0.1 mM Ca2’ but not that by 5 mM Ca2’ was sensitive to external Mg”+. Ryanodine (3 x 10m5M) applied during the plateau phase of the contraction due to 0.1 mM Ca2’ inhibited the tension after a transient potentiation but had no effect on that due to 5 mM Ca2’. Mg+ deficiency did not significantly affect the response to hypertonic 30 mM KC1 in Ca2+-containing PSS; i.e., the contraction was 58 t 4% with 1 mM Mg2+ (n = 6) and 70 t 7% with no Mg’ (n = 6, 100% = maximum contraction due to hypertonically added 60 mM KC1 in normal PSS).
0.A
A
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IN VASCULAR
SMOOTH
MUSCLE
As well as Mg+ deficiency, low temperature is considered to facilitate Ca2’-induced Ca2’ release (6), as demonstrated by the enhancement of caffeine-induced contractions (18,3Z) or by rapid cooling-induced contraction (23). If the Ca contraction was partly due to Ca2’-induced Ca2’ release, the contraction should be augmented by lowering the temperature. During the steady state of Ca contraction the abrupt fall of temperature of bathing fluid to 27°C increased the tension in the muscle contracted with 0.1 mM Ca2+, whereas the same procedure slightly decreased the tension developed by 5 mM Ca2+ (Fig. 3). The ryanodine modification of the SR Ca2+ release channel is dependent on the opening of the channel so that its effect on muscle contraction exhibits use-dependency (2, 11, 28). If Ca2’-induced Ca2’ release is involved in the Ca contraction, ryanodine should inhibit the contraction use- dependently. We therefore repeated the induction of the Ca contraction in the presence of ryanodine. Figure 4 shows the decrease of the Ca contraction by the presence of ryanodine at the first and second applications of 0.1 mM Ca2’ to depolarized aortas. The depression by ryanodine of the second contraction was larger than that of the first contraction during the first 3 min after the addition of Ca2’, indicating that usedependent inhibition appeared during the initial stage of the contraction. At later times the contraction upon the second application of Ca2’ did not differ from that of the first application. This means that the use-dependent effect developed gradually as the tension developed after the addition of Ca2’. Influence of Ca2+ concentration. In the next series of
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3. Effects of external Mg+ and lowering temperature of bathing fluid on 0.1 mM and 5 mM Ca*’ -induced contractions of guinea pig aorta preincubated in Ca*+- free isotonic KC1 solution. After we observed responses to Ca*+ in 1 mM M$+ solution, muscles were incubated in Mg’-free solution for 1 h until the next Ca contraction was elicited. During sustained phase of Ca contraction temperature was abruptly changed from 37°C to 27”C, or ryanodine (3 x 10m5 M) was added. FIG.
4
3 Time
5
10
(min)
FIG. 4. Use-dependent inhibition by ryanodine of Ca contraction on repeated induction of Ca contraction. Ca*’ at 0.1 mM was added to normally Ca*+- loaded muscles in Ca*+-free isotonic KC1 solution. Response was elicited once before addition of ryanodine (control) and twice in presence of 3 x 10s5 M ryanodine, which was applied 20 min before addition of Ca*+. Ryanodine-sensitive fraction is expressed by subtracting the response to first or second application of Ca*’ in presence of ryanodine from that in the control response. One hundred percent in ordinate represents maximal contraction due to hypertonitally added 60 mM KC1 to normal PSS. Squares, decrease of tension on first application of 0.1 mM Ca*’ in presence of 3 x low5 M ryanodine; circles, that on second application of Ca*‘. Each point represents mean k SE of 10 preparations. * Significantly different from ryanodinesensitive fraction in first contraction.
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cA2+
INFLUX
AND
CA2+ RELEASE
IN VASCULAR
SMOOTH
MUSCLE
experiments the influences of Ca2+ concentrations on ca2+ tension development by depolarized aortas and on the 0.1 mM OS mM 2S mM 1.0 mM inhibitory effect of ryanodine were examined. The protocol was the same as the aforementioned experiments, but the Ca2+ concentration was varied between 0.1 and 2.5 mM. Figure 5 shows the time course of tension development upon addition of each concentration of Ca2’. The contraction was always larger with higher Ca2+ concentration. However, the ryanodine-sensitive fraction in the contraction was larger at lower Ca2’ concentrations (0.1 and 0.5 mM) than at higher concentrations (1.0 and 2.5 mM). Furthermore, the ryanodine inhibition declined with time when 1.0 or 2.5 mM Ca2+ was added, whereas with 0.1 mM Ca2+ the ryanodine-sensitive fraction increased with time. lb0 260 250 6 & lb Figure 6 shows the relationship between 45Ca2+ uptake ‘%a Uptake (nmoVg mude) and ryanodine sensitivity of the Ca contraction for 5 min FIG. 6. Relationship between 45Ca2’ uptake and inhibition of the Ca after the addition of various concentrations of Ca2+. The ryanodine sensitivity is expressed in two ways: one is a contraction by ryanodine. 45Ca2+uptake and contraction were measured for 5 min with various Ca2’ concentrations as indicated above. Ryanodecrease of the contraction by ryanodine and the other dine inhibition of Ca contraction is expressed as decrease of tension by is a ratio of the ryanodine-sensitive fraction in the tenryanodine (open circles; 100% means maximum contraction due to sion development at 5 min. The ratio of ryanodinehypertonically added 60 mM KC1 to normal PSS) or ratio of inhibition sensitive fraction was inversely related to the 45Ca2+ by ryanodine (closed circles; 100% means tension development at 5 uptake. Thus, when the Ca2+ influx was larger, the con- min due to each concentration of Ca2’ in control muscles). tribution of the ryanodine-sensitive fraction to the total TABLE 1. Effects of Ca2+channel modulators on Ca2+contraction seemed to decrease. induced contraction of depolarized guinea pig aortas in Alteration of ryanodine-sensitive fraction in Ca contraction by Ca2+channel modulators. As mentioned above
the ryanodine-sensitive fraction of the Ca contraction is inversely related to Ca2+ concentration added, namely, to the amount of Ca2+ influx. Next, we tested whether the fraction varied when the Ca2’ influx through Ca2+ channels was modified by Ca2’ channel modulators. To decrease the Ca2+ influx, verapamil (1O-6 M) or cadmium (10e6 M) was applied 15 min before the reintroduction of 2.5 mM Ca2’, and to enhance the Ca2’ influx, CGPCL1 mM
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absence and presence of ryanodine Ca*+, mM
Drugs
Ryanodine Absent
Present
%Inhibition by Ryanodine
n
Control 31.7k4.4 11.3t2.0* 62.6k4.9 10 CGP-28392 43.6k4.6 26.3t3.5’t 42.4k6.27 8 2.5 Control 89.9k4.0 74.3k8.0 18.4k7.1 7 Verapamil 42.3k7.57 23.7*5.9*? 44.8t9.0-f 6 Cadmium 33.7k10.27 3.5+2.8*7 94.7+3.8-f 6 Values are means t SE. Values in left 2 columns represent tension development at 5 min after addition of Ca2’ (100% = maximum contraction due to 60 mM KC1 added in normal PSS). CGP-28392 ( 10m5M), verapamil ( 10s6 M), and cadmium ( 10B5 M) were added when external medium was changed to 0-Ca iso-K solution. When present, ryanodine (3 x 10N5 M) was added 20 min before reintroduction of Ca2+. * Significantly different from contraction in the absence of ryanodine (paired t test, P < 0.05). t Significantly different from control (nonpaired t test, P < 0.05). 0.1
28392 (25) was added 15 min before 0.1 mM Ca2+. The tension development at 5 min after Ca2’ application under each condition is summarized in Table 1. Verapamil and cadmium decreased the contraction, but both substances augmented the ratio of the ryanodine-sensitive fraction. On the other hand, CGP-28392 increased the contraction while decreasing the ratio of the ryanodine-sensitive fraction. DISCUSSION
(min)
. Tension development of depc11; arized guinea pig aorta when the Ca2’ concentration added at 0 min was varied. Concentrations of Ca2’ given are shown in each panel. Ordinate represents relative tension of maximum contraction due to hypertonically added 60 mM KC1 to normal PSS. Closed circles, control; open circles, 3 x 10S5 M ryanodine, which was applied 20 min before addition of Ca2’. Symbols for statistical significance are omitted from figure. Data are expressed as mean * SE of 6-12 preparations.
The SR can handle intracellular Ca2+ through two functions: one is a buffering of Ca2’ that enters the cells from extracellular space and the other is a Ca2’ release. With regard to the former, the state of filling of the store may alter the amount of Ca2’ available for the binding to calmodulin. If the SR is poorly loaded with Ca2’, a large portion of Ca2’ entering the cell would be taken up
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H1468
cA2+
INFLUX
AND
cA2+
RELEASE
by the SR; thus less Ca2’ is available for a contraction. On the contrary, if the SR is saturated with Ca2+, then Ca2’ from extracellular fluid would bypass the SR, resulting in a potentiation of a contraction (“buffer barrier” function, Ref. 34). On the other hand, Ca2’ release from the SR depends on the filling of Ca2’ in the organelle (14). Therefore, in a muscle with a fully Ca2+-loaded SR, both a Ca2+ release and a circumventing Ca2+ to a great extent may enhance the contraction, whereas in a Ca2+-depleted muscle the tension development may be small because of the less Ca2+ release and the absorption of Ca2’ to the SR. Thus the difference between the responses to Ca2’ in Ca2+-loaded muscles and Ca2+-depleted ones could come from these factors. If the Ca2’ buffering by the SR is more important in regulating the cytoplasmic Ca2’ concentration than the Ca2’ release, the treatment with ryanodine should potentiate the contraction, since the SR may not be able to retain Ca2’ in the presence of ryanodine (9,10,17), so that Ca2+ across the sarcolemma would bypass the SR. This was observed in some experiments (3, 16). In the present study, however, the treatment with ryanodine instead decreased the contractions in both Ca2+-loaded and Ca2+-depleted muscles. This suggests that a change in Ca2’ buffering by either Ca2+loading or ryanodine treatment did not exert a substantial influence on the Ca contraction observed in this study. We can therefore say that the greater response to Ca2’ in Ca2’-loaded muscles is mainly caused by greater Ca2+ release. The contraction induced by 0.1 mM Ca2’ was potentiated by a M$+ deficiency in the external medium. Because the tension development induced by 30 mM KC1 in normal PSS or by an introduction of 5 mM Ca2’ in OCa iso-K solution was not affected by the removal of external Mg+, the possible involvement of a change in Ca2+ influx or Ca2+ sensitivity of contractile apparatus caused by M$+ deficiency had a negligible influence on the augmented response to 0.1 mM Ca2’ in depolarized muscles. The potentiation similar to a potentiation of caffeine-induced contraction by Mg+-deficient solution (18, 30, 32) suggests that this was due to the enhanced Ca2’-induced Ca2’ release. The contraction due to 0.1 mM Ca2’ was also enhanced by lowering the temperature of the medium, whereas the contraction due to 5 mM Ca2+ was decreased by this procedure. This is also consistent with the observations that the low temperature enhances a contraction due to Ca2+-induced Ca2’ release (6, 23, 32). Furthermore, ryanodine exhibited use-dependent inhibition on the contraction, which means that opening Ca2+ release channels augments the action of ryanodine (2). These findings support the involvement of Ca2+-induced Ca2+ release in the Ca contraction. We can therefore conclude that the Ca2’ influx through voltage-dependent Ca2+ channels triggered the release of Ca2+ from the SR. The Ca2+ release by caffeine from SR of vascular smooth muscle is transient either in Ca2+-containing or Ca2+-free medium (4, 14, 32). This means that the SR cannot be reprimed with Ca2+ in the presence of caffeine even if Ca2+ is present in the cytoplasm. Ryanodine inhibited the entire course of Ca contraction over a 20min observation period when the added Ca2’ was 0.1 or
IN
VASCULAR
SMOOTH
MUSCLE
0.5 mM. Therefore, unlike the Ca2+ release by caffeine, the Ca2+ influx-induced Ca2+ release is likely to continuously occur as long as Ca2+ influx persists to refill the SR When 0.1 mM Ca2’ was applied to Ca2+-depleted muscle, the contraction at 2 min in the absence or presence of ryanodine was 10e5 M (20,24) or 2 X 10B6 M (5) in isolated skeletal or cardiac SR vesicles. In addition, the concentration of cytoplasmic Ca2+ required to inactivate the Ca2+-induced Ca2’ release mechanism in vascular smooth muscle has not been examined. Therefore it remains unclear whether the inactivation of the mechanism can occur with a physiological concentration of Ca2’ in intact vascular
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CA*+
INFLUX
AND CA*+ RELEASE
smooth muscles. Another possibility regarding the smaller ryanodinesensitive fraction when Ca2+ influx was high is that Ca2’ which entered cells through Ca2+ channels was sufficient to induce a maximum contraction by itself and surpassed the involvement of the Ca2+-induced Ca2’ release. As shown in Fig. 5, however, 0.5 and 1.0 mM Ca2’ caused the same magnitude of steady contraction, but the inhibition by ryanodine was larger with lower Ca2+. This suggests that Ca2’ -induced Ca2’ release plays a minor role in 1 mM Ca2’-induced contraction compared with that by 0.5 mM Ca2’ despite the same tension development. Himpens et al. (8) measured the intracellular Ca2’ signal by fi.ua-2 when Ca2+ was applied to depolarized rabbit pulmonary artery. The application of 0.65 mM Ca2’ caused an almost maximal contraction, as did 1 mM Ca2+, but the elevation of intracellular Ca2’ was slightly less with 0.65 mM Ca2+ than with 1 mM Ca2’. Similarly, in our study, the 45Ca2+ influx from extracellular fluid was less with 0.5 mM Ca2’ than with 1.0 mM Ca2+. Therefore the tension development of depolarized muscles due to >l mM Ca2+ may not be proportional to intracellular Ca2+ concentration. A further study with an intracellular Ca2+ indicator is needed to determine whether or how much Ca2’ release occurs when a high Ca2’ concentration is used. In any case the present data suggest that the inactivation of Ca2’ release by cytoplasmic Ca2+, if any, occurs at the Ca2+ concentration which by itself causes nearly maximal contraction with no help of Ca2+ release. In conclusion, this study showed that if Ca2’ influx via voltage-dependent Ca2+ channels is in the physiological range, Ca2’-induced Ca2’ release exerts a significant contribution to contractions. We thank Dr. J. L. Sutko for careful review of the manuscript. This work was partly supported by Ministry of Education, Science and Culture of Japan Grant 02660312. Address for reprint requests: K. Ito, Dept. of Veterinary Pharmacology, Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan. Received 5 November 1990; accepted in final form 17 June 1991.
SMOOTH
H1469
MUSCLE
C14,1983. 8. HIMPENS,
B., G. MATTHIJS, AND A. P. SOMLYO. Desensitization to cytoplasmic Ca*+ and Ca*+ sensitivities of guinea-pig ileum and rabbit pulmonary artery smooth muscle. J. Physiol. Land. 413: 489-
503,1989. 9. HWANG,
K. S., AND C. VAN BREEMEN. Ryanodine modulation of 45Ca efflux and tension in rabbit aortic smooth muscle. Pfluegers
Arch. 10. IINO,
408: 343-350,1987. M., T. KOBAYASHI,
AND M. ENDO. Use of ryanodine for functional removal of the calcium store in smooth muscle cells of the guinea-pig. Biochem. Biophys. Res. Commun. 152: 417-422, 1988.
11. ITO, K., T. IKEMOTO, S. AOKI, AND M. OTA. Effects of ryanodine and 9,21didehydroryanodine on caffeine-induced contraction of rat and guinea pig aortae. Jpn. J. Pharmacol. 51: 531-538, 1989. 12. ITO, K., T. IKEMOTO, AND S. TAKAKURA. Evaluation of contribution of calcium influx-induced calcium release to the contraction of intact vascular smooth muscle. In: Biosignalling in Cardiac and Vascular Systems, edited by M. Fujiwara, S. Narumiya, and S. Miwa. Oxford, UK: Pergamon, 1989, p. 228-231. 13. ITO, K., S. TAKAKURA, K. SATO, AND J. L. SUTKO. Ryanodine inhibits the release of calcium from intracellular stores in guineapig aortic smooth muscles. Circ. Res. 58: 730-734, 1986. 14. ITOH, T., H. KURIYAMA, AND H. SUZUKI. Excitation-contraction coupling in smooth muscle cells of the guinea-pig mesenteric artery. J. Physiol. 15. ITOH, T.,
Land.
321: 513-535, 1981. AND H. KURIYAMA.
H. UENO, release mechanism based on contractions muscles. Experientia
Calcium-induced calcium in vascular smooth muscles-assessments evoked in intact and saponin-treated skinned
Base1 41: 989-996, 1985. G., AND J. L. FRESLON.
Effects of ryanodine on tension development in rat aorta and mesenteric resistance vessels. Br. J. Pharmacol. 95: 605-613, 1988. 17. KANMURA, Y., L. MISSIAEN, L. RAEYMAEKERS, AND R. CASTEELS. Ryanodine reduces the amount of calcium in intracellular stores of smooth-muscle cells of the rabbit ear artery. Pfluegers Arch. 413: 16. JULOU-SCHAEFFER,
153-159, 18. KARAKI,
1988.
H., H. Y. AHN, AND N. URAKAWA. Caffeine-induced contraction in vascular smooth muscle. Arch. Int. Pharmacodyn.
285: 60-71,1987. 19. KARAKI, H., AND
G. B. WEISS. Alterations in high and low affinity binding of 45Ca in rabbit aortic smooth muscle by norepinephrine and potassium after exposure to lanthanum and low temperature.
J. Pharmacol. 20. KIRINO, Y.,
Exp.
Ther.
211: 86-92,
1979.
M. OSAKABE, AND H. SHIMIZU. Ca*+-induced Ca*’ release from fragmented sarcoplasmic reticulum: Ca*+-dependent passive Ca*’ efflux. J. Biochem. 94: 1111-1118, 1983. 21. KITAZAWA, T., S. KOBAYASHI, K. HORIUTI, A. V. SOMLYO, AND A. P. SOMLYO. Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca*‘. J. Biol. Chem. 264: 5339-5342, 1989. 22. LEIJTEN,
REFERENCES H. Y., AND H. KARAKI. Inhibitory effects of procaine on contraction and calcium movement in vascular and intestinal smooth muscles. Br. J. Pharmacol. 94: 789-796, 1988. 2. AOKI S., AND K. ITO. Time- and use-dependent inhibition by ryanodine of caffeine-induced contraction of guinea-pig aortic smooth muscle. Biochem. Biophys. Res. Commun. 154: 219-226, 1. AHN,
1988. 3. ASHIDA, T., J. SCHAEFFER, W. F. GOLDMAN, P. BLAUSTEIN. Role of sarcoplasmic reticulum
J. B. WADE, AND M. in arterial contraction: comparison of ryanodine’s effect in a conduit and a muscular artery. Circ. Res. 62: 854-863, 1988. 4. CASTEELS, R., K. KITAMURA, H. KURIYAMA, AND H. SUZUKI. Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery. J. Physiol. Land. 271: 63-79, 1977. 5. CHAMBERLAIN, B. K., P. VOLPE, AND S. FLEISCHER. Calciuminduced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics. J. Biol. Chem. 259: 75407546, 1984.
6. ENDO, M. Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57: 71-108,1977. 7. FABIATO, A. Calcium-induced release sarcoplasmic reticulum. Am. J. Physiol.
IN VASCULAR
of calcium from the cardiac 245 (Cell Physiol. 14): Cl-
P. A. A., AND C. VAN BREEMEN. The effects of caffeine on the noradrenaline-sensitive calcium store in rabbit aorta. J.
Physiol. Land. 23. MAGARIBUCHI,
357: 327-339, T., Y. ITO,
1984. AND
H. KURIYAMA. Effects of rapid cooling on the mechanical and electrical activities of smooth muscles of guinea pig stomach and taenia coli. J. Gen. Physiol. 61: 323-
341,1973. 24. MEISSNER,
G., AND J. S. HENDERSON. Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca*’ and is modulated by M$‘, adenine nucleotide, and calmodulin. J. Biol.
Chem. 262: 3065-3073,1987. 25. MIKKELSEN, E. O., AND
N. C. B. NYBORG. Comparison of the effects of the vasoconstrictive dihydropyridines BAY K 8644 and CGP 28392 on isolated rat aorta: different sensitivities to ultraviolet radiation. J. Cardiovasc. Phurmacol. 8: 476-482, 1986. 26. NISHIMURA, J., M. KOLBER, AND C. VAN BREEMEN. Norepinephrine and GTP-7-S increase myofilament Ca*’ sensitivity in a-toxin permeabilized arterial smooth muscle. Biochem. Biophys. Res. Commun. 157: 677-683, 1988. 27. PUTNEY, J. W., JR. A model for receptor-regulated calcium Cell Calcium 7: 1-12, 1986. 28. ROUSSEAU, E., J. S. SMITH, AND G. MEISSNER. Ryanodine
entry.
modifies conductance and gating behavior of single Ca”’ release channel.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 12, 2019.
H1470
CA2+
Am. J. Physiol. 253 (Cell Physiol. 29. ROEGG, J. C., AND R. J. PAUL.
INFLUX
AND CA2+ RELEASE
22): C364-C368,
1987.
Vascular smooth muscle. Calmodulin and cyclic AMP-dependent protein kinase alter calcium sensitivity in porcine carotid skinned fibers. Circ. Res. 50: 394-399,
1982. 30. SAIDA, K. Intracellular Ca release Gen. Physiol. 80: 191-202, 1982. 31. SAIDA, K., AND C. VAN BREEMEN.
adrenoceptor-mediated Physiol.
84: 307-318,
in skinned smooth muscle. J.
Cyclic AMP modulation of arterial smooth muscle contraction. J. Gen.
1984.
IN VASCULAR
SMOOTH
MUSCLE
K., H, OZAKI, AND H. KARAKI. Multiple effects of caffeine on contraction and cytosolic free Ca2+ levels in vascular smooth muscle of rat aorta. Nuunyn-Schmiedeberg’s Arch. Pharmacol. 338:
32. SATO,
443-448,1988. 33. SUTKO, J.
L., K. ITO, AND J. L. KENYON. Ryanodine: a modifier of sarcoplasmic reticulum calcium release in striated muscle. Fed-
eration Proc. 44: 2984-2988, 1985. 34. VAN BREEMEN, C., C. CAUVIN, A. JOHNS, P. LEIJTEN, AND YAMAMOTO. Ca2+ regulation of vascular smooth muscle. Federation Proc. 45: 2746-2751,1986.
H.
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ITO,
TAKAAKI
IKEMOTO,
AND
SACHIKO
TAKAKURA
Department of Veterinary Pharmacology, Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan
lease of Ca2+ from SR in vascular smooth muscles (9,10, 13). Unlike procaine (1) or caffeine (18, 22, 32), ryanodine acts selectively on the SR because it does not directly affect either transmembrane Ca2+ influx or the contractile machinery (17, 33). Therefore the sensitivity isotonic KC1 solution induced a contraction (Ca contraction), of a contraction to ryanodine can be interpreted as the a part of which dependedon preloading the intracellular stores with Ca*’ and which was sensitive to 3 x 10m5 M ryanodine. presence of a component associated with Ca2’ release Because‘%a*+ influx from the external fluid upon the addition from the SR. We have used ryanodine as a tool to analyze Ca2+ chanof Ca*’ was not modified either by the state of filling of the how Ca2’ influx through voltage-dependent Ca*’ store or by the presence of ryanodine, the ryanodine- nels causes Ca2+ release in intact guinea pig aortic sensitive component of the contraction could be attributed to smooth muscles depolarized by high K+. ITO, KATSUAKI, TAKAAKI IKEMOTO, AND SACHIKO TAKACa*’ release in conKURA. Involvement of Ca*+ influx-induced tractions of intact vascular smooth muscles. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1464-H1470,1991.-Application of Ca*+ (0.1-2.5 mM) to guinea pig aortas incubated in Ca*‘-free
the Ca*+influx-induced Ca*’ releasefrom the Ca*’ store. Supporting the possibility of involvement of Ca*‘-induced Ca*’ release,the Ca contraction due to 0.1 mM Ca*’ was enhanced either by decreasingMg2+in the mediumor by low temperature. The ratio of the ryanodine-sensitive fraction in the Ca contraction was inversely related to the concentration of Ca*’ added and also to the extent of 45Ca2+ influx. When the Ca*’ influx wasdecreasedby verapamil or cadmium,the ratio of ryanodinesensitive fraction increased. On the contrary, an increase of Ca*’ influx by CGP-28392 decreasedthe ratio. These results suggestthat Ca*’ influx at a physiological level triggers Ca*’ releasefrom the Ca*’ store, resulting in the amplification of contractile force.
METHODS
Preparations. Guinea pigs weighing 250-450 g were stunned by a blow on the neck, and the thoracic aortas were isolated. Rectangular strips of aorta were made from aortic rings of 2-3 mm in width. The muscles were suspended in an organ bath containing 5 ml physiological saline solution (PSS; in mM: 136.8 NaCl, 5.4 KCl, 2.5 CaC12, 0.5 MgC12, 11.9 NaHC03, and 5.5 dextrose, pH 7.3-7.4) gassed with 95% 02-5% CO2 (37°C unless otherwise stated). They were equilibrated in PSS for 1 h before experiments, and a basal tension of 1 g was sarcoplasmicreticulum; calcium channel; depolarized muscle; maintained. guineapig aorta; ryanodine Tension experiments. The Ca2’-induced contraction (termed as Ca contraction) was elicited by reintroduction of Ca2+ at various concentrations to muscles preincuIT IS NOW WIDELY ACCEPTED that the Ca2+-induced bated in nominally Ca2’- free isotonic KC1 solution (Orelease of Ca2+ from sarcoplasmic reticulum (SR) can Ca iso-K solution; in mM: 142.2 KCl, 0.5 MgC12, 11.9 occur in every type of muscle (6). This can be demonNaHCOs, and 5.5 dextrose) for 20 min. Before exposure strated experimentally as a caffeine-induced contraction. to 0-Ca iso-K solution, the intracellular Ca2+ store of the Many vascular smooth muscles respond to caffeine with muscle was loaded with Ca2’ to different degrees, i.e., a transient contraction in intact or skinned preparations, normal loading, excess loading, or depletion of Ca2+ from suggesting the existence of a Ca2+-induced Ca2’ release the store. To normally load the store, the muscles were mechanism (14, 15). However, the manifestation of caf- exposed to normal PSS for 15 min after lo-min preinfeine-induced contraction does not necessarily mean that cubation in Ca2+-free PSS. If the store was loaded with the Ca2+-induced Ca2+ release mechanism plays a signifCa2’ in 2.5 mM Ca2+-PSS for more than 5 min, a conicant role under physiological conditions in intact vas- stant Ca contraction was obtained. Unless specified, the cular smooth muscles. To assess whether this mechanism muscles were loaded with Ca2+ in this way. Excess loadis physiologically important in a given muscle, the ability ing was achieved by exposing the aorta to hypertonically of Ca2+ influx across the plasma membrane to cause Ca2’ added 60 mM KC1 in the presence of 2.5 mM Ca2+ for 15 release from the SR must be demonstrated. This is an min until the external medium was switched to O-Ca isoimportant issue because various transmitters and hor- K solution. The effectiveness of this procedure was conmones cause both transmembrane Ca2+ influx and intrafirmed by the experiment that this procedure potentiated cellular Ca2+ release after activation of the receptors in the contraction due to norepinephrine applied in Ca2+vascular smooth muscles. free PSS to 170% of that after normal loading. Ca2’ Ryanodine ultimately abolishes the Ca2+-induced re- deprivation was achieved by consecutive exposures to H1464
0363-6135/91
$1.50
Copyright 0 1991 the American Physiological Society
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cA2+
INFLUX
AND
CA2+
RELEASE
norepinephrine ( 10m6M) or caffeine (30 mM) in Ca2’free PSS until the phasic contraction due to norepinephrine or caffeine was abolished. 45Ca2+uptake experiments. Cellular 45Ca2+ uptake into aortic smooth muscles was measured by the cold lanthanum washing method (19). A thoracic aorta was divided into four strips, each of which was 8-12 mg wet wt. Muscles were loaded with 1 g of tension in a circular direction and equilibrated in normal tris(hydroxymethyl)aminomethane (Tris)-PSS containing (in mM) 136.8 NaCl, 5.4 KCl, 2.5 CaC12, 0.5 MgC12, 5.5 dextrose, and 5.0 Tris-HCl (pH 7.4) and gassed with 100% O2 for 60 min at 37OC. During the equilibration period muscles were conditioned with hypertonic 65.4 mM KC1 for between 15 and 30 min. The muscles were transferred to Ca2+-free isotonic KC1 solution (buffered with 5 mM Tris). Twenty minutes later 0.1, 0.5, 1.0 or 2.5 mM Ca2’ with 45Ca2+ (1 &i/ml) was applied. Five or 30 min later the muscles were transferred to La3+ solution (in mM: 80.8 LaC13, 5.5 dextrose, and 5 Tris, pH 6.8) cooled to below 2°C to displace the extracellular Ca2+ during the 60-min immersion. After that, the muscle was blotted between two filter papers (Whatman no. 2) under a l5g/cm2 weight for 10 s, and the wet weight was measured on a torsion balance. The residual radioactivity was counted as cellular 45Ca2+ uptake. Reagents. The following drugs were used: ryanodine (lot 704RWP-1, S. B. Penick, Lyndhurst, NJ), norepinephrine (Sigma, St. Louis, MO), verapamil (Eisai, Tokyo, Japan), and CGP-28392 (Ciba-Geigy, Basel, Switzerland). CGP-28392 was dissolved in dimethyl sulfoxide; other drugs were dissolved in distilled water. Statistics. Data are expressed as mean t SE. Significance was considered at the level of P < 0.05.
IN VASCULAR
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Ca2+-induced contraction of guinea pig aorta preincubated in Ca2+-free, isotonic KC1 solution. Before addition of Ca2’ intracellular Ca2+ store was overloaded or normally loaded with Ca2’ or depleted of Ca2’ (see METHODS). At 0 min 0.1 mM Ca2’ was added. One hundred percent in the ordinate represents maximum contraction induced by hypertonically added 60 mM KC1 to normal physiological saline solution (PSS). Filled circles, control; open circles, in the presence of 3 X lo-” M ryanodine, which was applied 20 min before addition of Ca2’. Each point represents mean t SE of 6-10 preparations. * Significantly different from control (P < 0.05). FIG.
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Ca2’ to vascular smooth muscle incubated in a Ca2+-free high-K+ solution causes transmembrane Ca2’ influx and induces a contraction. A simplistic view of this event is that Ca2+ coming from the external milieu directly activates the contractile machinery. However, the Ca2’ can also trigger the release of Ca2’ from SR. Our previous papers (12, 13) showed that a fraction of the Ca2+induced contraction of depolarized guinea pig aorta was sensitive to ryanodine, suggesting that Ca2+ release was involved in the contraction. In this study we analyzed the detailed nature of the Ca contraction. Figure 1 shows the time course of 0.1 mM Ca2+-induced contraction over a lo-min period in guinea pig aortas, which had been preincubated in 0-Ca iso-K solution, under three states of Ca2+ loading of the Ca2’ store, i.e., overloading, normal loading, or depletion of Ca2+. As shown in Fig. 1, the contraction in Ca2+-overloaded muscles rose rapidly with a large amplitude upon the addition of 0.1 mM Ca2+. On the other hand, the tension development in Ca2+ -depleted muscles was very slow and small. The rate of rise and the amplitude of the Ca contraction in normally loaded muscles were intermediate. Ryanodine (3 x 10m5 M) inhibited the development
k&ed
2
RESULTS
Dependence of Ca2+-induced contraction of depolarized aorta on Ca2+ loading of Ca2+ store. The application of
H1465
MUSCLE
30 min
-il
l .*.*.v l .*.*.v l .*.v.* v.*.v 5v.v v.*.v l .v.v .*.*.v ll .*.*.v v.*.*.* y normally loaded > Ca2+-depleted muscle. Especially, in Ca2+-depleted muscle, ryanodine did not significantly decrease the Ca contraction during first 5 min. 45Ca2+uptake into Ca2+-loaded muscles and Ca2+-depleted muscles. With regard to the different magnitudes
of responses of depolarized aorta to added Ca2+, it was possible that the degree of Ca2’ influx was different between Ca2+-loaded and Ca2’-depleted muscles, since Putney (27) postulated that Ca2’ influx depended on the emptying of Ca2+ stores in some cells. To test this, 45Ca2+ uptake into Ca2’ -overloaded and Ca2+-depleted muscles was measured. Figure 2 shows the 45Ca2+ uptake into depolarized aortas during a 0.1 mM Ca2+-induced contraction. The 45Ca2+ influx in the absence of ryanodine was not different between Ca2’-loaded and Ca2+-depleted muscles. Furthermore, ryanodine did not affect the 45Ca
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H1466
CA*+
INFLUX
AND
CA*+
RELEASE
influx for 5 or 30 min after the addition of Ca2’. These results suggest that the greater response to Ca2’ of the Ca2+-loaded muscle was not due to augmented Ca2’ influx and also that the greater sensitivity to ryanodine in such muscle was not due to inhibition of Ca2’ influx by ryanodine. Some characteristics of the Ca contraction. The above results suggest that Ca2+- induced Ca2’ release is involved in the contraction induced by the addition of 0.1 mM Ca2’ into 0-Ca iso-K solution. Three further tests were performed to determine whether the Ca contraction due to 0.1 mM Ca2’ had some characteristics of Ca2’-induced Ca2+ release. Mg2+ modulates the Ca2’ -induced Ca2’ release mechanism (6). In fact, contractions of smooth muscles due to caffeine were reportedly potentiated by incubation of muscle in Mg’-deficient solution (18, 30, 32). If the Ca2’-induced Ca2’ release mechanism operates in the Ca contraction elicited by the protocol used in this study, the contraction should be enhanced by incubation with Mg’-free PSS. Figure 3 shows that this is the case. When the muscle was equilibrated in Mg+-free PSS for >l h and then exposed to Ca2’- and Mg+-free isotonic KC1 solution, the contraction induced by 0.1 mM Ca2+ was larger compared with that preincubated with 1 mM Mg+, whereas the contraction induced by 5 mM Ca2’ was the same or slightly depressed in Mg2’-deficient solution compared with Mg’-containing PSS. Thus the contraction of depolarized aorta induced by 0.1 mM Ca2’ but not that by 5 mM Ca2’ was sensitive to external Mg”+. Ryanodine (3 x 10m5M) applied during the plateau phase of the contraction due to 0.1 mM Ca2’ inhibited the tension after a transient potentiation but had no effect on that due to 5 mM Ca2’. Mg+ deficiency did not significantly affect the response to hypertonic 30 mM KC1 in Ca2+-containing PSS; i.e., the contraction was 58 t 4% with 1 mM Mg2+ (n = 6) and 70 t 7% with no Mg’ (n = 6, 100% = maximum contraction due to hypertonically added 60 mM KC1 in normal PSS).
0.A
A
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A WC
IN VASCULAR
SMOOTH
MUSCLE
As well as Mg+ deficiency, low temperature is considered to facilitate Ca2’-induced Ca2’ release (6), as demonstrated by the enhancement of caffeine-induced contractions (18,3Z) or by rapid cooling-induced contraction (23). If the Ca contraction was partly due to Ca2’-induced Ca2’ release, the contraction should be augmented by lowering the temperature. During the steady state of Ca contraction the abrupt fall of temperature of bathing fluid to 27°C increased the tension in the muscle contracted with 0.1 mM Ca2+, whereas the same procedure slightly decreased the tension developed by 5 mM Ca2+ (Fig. 3). The ryanodine modification of the SR Ca2+ release channel is dependent on the opening of the channel so that its effect on muscle contraction exhibits use-dependency (2, 11, 28). If Ca2’-induced Ca2’ release is involved in the Ca contraction, ryanodine should inhibit the contraction use- dependently. We therefore repeated the induction of the Ca contraction in the presence of ryanodine. Figure 4 shows the decrease of the Ca contraction by the presence of ryanodine at the first and second applications of 0.1 mM Ca2’ to depolarized aortas. The depression by ryanodine of the second contraction was larger than that of the first contraction during the first 3 min after the addition of Ca2’, indicating that usedependent inhibition appeared during the initial stage of the contraction. At later times the contraction upon the second application of Ca2’ did not differ from that of the first application. This means that the use-dependent effect developed gradually as the tension developed after the addition of Ca2’. Influence of Ca2+ concentration. In the next series of
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0 mM
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3. Effects of external Mg+ and lowering temperature of bathing fluid on 0.1 mM and 5 mM Ca*’ -induced contractions of guinea pig aorta preincubated in Ca*+- free isotonic KC1 solution. After we observed responses to Ca*+ in 1 mM M$+ solution, muscles were incubated in Mg’-free solution for 1 h until the next Ca contraction was elicited. During sustained phase of Ca contraction temperature was abruptly changed from 37°C to 27”C, or ryanodine (3 x 10m5 M) was added. FIG.
4
3 Time
5
10
(min)
FIG. 4. Use-dependent inhibition by ryanodine of Ca contraction on repeated induction of Ca contraction. Ca*’ at 0.1 mM was added to normally Ca*+- loaded muscles in Ca*+-free isotonic KC1 solution. Response was elicited once before addition of ryanodine (control) and twice in presence of 3 x 10s5 M ryanodine, which was applied 20 min before addition of Ca*+. Ryanodine-sensitive fraction is expressed by subtracting the response to first or second application of Ca*’ in presence of ryanodine from that in the control response. One hundred percent in ordinate represents maximal contraction due to hypertonitally added 60 mM KC1 to normal PSS. Squares, decrease of tension on first application of 0.1 mM Ca*’ in presence of 3 x low5 M ryanodine; circles, that on second application of Ca*‘. Each point represents mean k SE of 10 preparations. * Significantly different from ryanodinesensitive fraction in first contraction.
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cA2+
INFLUX
AND
CA2+ RELEASE
IN VASCULAR
SMOOTH
MUSCLE
experiments the influences of Ca2+ concentrations on ca2+ tension development by depolarized aortas and on the 0.1 mM OS mM 2S mM 1.0 mM inhibitory effect of ryanodine were examined. The protocol was the same as the aforementioned experiments, but the Ca2+ concentration was varied between 0.1 and 2.5 mM. Figure 5 shows the time course of tension development upon addition of each concentration of Ca2’. The contraction was always larger with higher Ca2+ concentration. However, the ryanodine-sensitive fraction in the contraction was larger at lower Ca2’ concentrations (0.1 and 0.5 mM) than at higher concentrations (1.0 and 2.5 mM). Furthermore, the ryanodine inhibition declined with time when 1.0 or 2.5 mM Ca2+ was added, whereas with 0.1 mM Ca2+ the ryanodine-sensitive fraction increased with time. lb0 260 250 6 & lb Figure 6 shows the relationship between 45Ca2+ uptake ‘%a Uptake (nmoVg mude) and ryanodine sensitivity of the Ca contraction for 5 min FIG. 6. Relationship between 45Ca2’ uptake and inhibition of the Ca after the addition of various concentrations of Ca2+. The ryanodine sensitivity is expressed in two ways: one is a contraction by ryanodine. 45Ca2+uptake and contraction were measured for 5 min with various Ca2’ concentrations as indicated above. Ryanodecrease of the contraction by ryanodine and the other dine inhibition of Ca contraction is expressed as decrease of tension by is a ratio of the ryanodine-sensitive fraction in the tenryanodine (open circles; 100% means maximum contraction due to sion development at 5 min. The ratio of ryanodinehypertonically added 60 mM KC1 to normal PSS) or ratio of inhibition sensitive fraction was inversely related to the 45Ca2+ by ryanodine (closed circles; 100% means tension development at 5 uptake. Thus, when the Ca2+ influx was larger, the con- min due to each concentration of Ca2’ in control muscles). tribution of the ryanodine-sensitive fraction to the total TABLE 1. Effects of Ca2+channel modulators on Ca2+contraction seemed to decrease. induced contraction of depolarized guinea pig aortas in Alteration of ryanodine-sensitive fraction in Ca contraction by Ca2+channel modulators. As mentioned above
the ryanodine-sensitive fraction of the Ca contraction is inversely related to Ca2+ concentration added, namely, to the amount of Ca2+ influx. Next, we tested whether the fraction varied when the Ca2’ influx through Ca2+ channels was modified by Ca2’ channel modulators. To decrease the Ca2+ influx, verapamil (1O-6 M) or cadmium (10e6 M) was applied 15 min before the reintroduction of 2.5 mM Ca2’, and to enhance the Ca2’ influx, CGPCL1 mM
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absence and presence of ryanodine Ca*+, mM
Drugs
Ryanodine Absent
Present
%Inhibition by Ryanodine
n
Control 31.7k4.4 11.3t2.0* 62.6k4.9 10 CGP-28392 43.6k4.6 26.3t3.5’t 42.4k6.27 8 2.5 Control 89.9k4.0 74.3k8.0 18.4k7.1 7 Verapamil 42.3k7.57 23.7*5.9*? 44.8t9.0-f 6 Cadmium 33.7k10.27 3.5+2.8*7 94.7+3.8-f 6 Values are means t SE. Values in left 2 columns represent tension development at 5 min after addition of Ca2’ (100% = maximum contraction due to 60 mM KC1 added in normal PSS). CGP-28392 ( 10m5M), verapamil ( 10s6 M), and cadmium ( 10B5 M) were added when external medium was changed to 0-Ca iso-K solution. When present, ryanodine (3 x 10N5 M) was added 20 min before reintroduction of Ca2+. * Significantly different from contraction in the absence of ryanodine (paired t test, P < 0.05). t Significantly different from control (nonpaired t test, P < 0.05). 0.1
28392 (25) was added 15 min before 0.1 mM Ca2+. The tension development at 5 min after Ca2’ application under each condition is summarized in Table 1. Verapamil and cadmium decreased the contraction, but both substances augmented the ratio of the ryanodine-sensitive fraction. On the other hand, CGP-28392 increased the contraction while decreasing the ratio of the ryanodine-sensitive fraction. DISCUSSION
(min)
. Tension development of depc11; arized guinea pig aorta when the Ca2’ concentration added at 0 min was varied. Concentrations of Ca2’ given are shown in each panel. Ordinate represents relative tension of maximum contraction due to hypertonically added 60 mM KC1 to normal PSS. Closed circles, control; open circles, 3 x 10S5 M ryanodine, which was applied 20 min before addition of Ca2’. Symbols for statistical significance are omitted from figure. Data are expressed as mean * SE of 6-12 preparations.
The SR can handle intracellular Ca2+ through two functions: one is a buffering of Ca2’ that enters the cells from extracellular space and the other is a Ca2’ release. With regard to the former, the state of filling of the store may alter the amount of Ca2’ available for the binding to calmodulin. If the SR is poorly loaded with Ca2’, a large portion of Ca2’ entering the cell would be taken up
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H1468
cA2+
INFLUX
AND
cA2+
RELEASE
by the SR; thus less Ca2’ is available for a contraction. On the contrary, if the SR is saturated with Ca2+, then Ca2’ from extracellular fluid would bypass the SR, resulting in a potentiation of a contraction (“buffer barrier” function, Ref. 34). On the other hand, Ca2’ release from the SR depends on the filling of Ca2’ in the organelle (14). Therefore, in a muscle with a fully Ca2+-loaded SR, both a Ca2+ release and a circumventing Ca2+ to a great extent may enhance the contraction, whereas in a Ca2+-depleted muscle the tension development may be small because of the less Ca2+ release and the absorption of Ca2’ to the SR. Thus the difference between the responses to Ca2’ in Ca2+-loaded muscles and Ca2+-depleted ones could come from these factors. If the Ca2’ buffering by the SR is more important in regulating the cytoplasmic Ca2’ concentration than the Ca2’ release, the treatment with ryanodine should potentiate the contraction, since the SR may not be able to retain Ca2’ in the presence of ryanodine (9,10,17), so that Ca2+ across the sarcolemma would bypass the SR. This was observed in some experiments (3, 16). In the present study, however, the treatment with ryanodine instead decreased the contractions in both Ca2+-loaded and Ca2+-depleted muscles. This suggests that a change in Ca2’ buffering by either Ca2+loading or ryanodine treatment did not exert a substantial influence on the Ca contraction observed in this study. We can therefore say that the greater response to Ca2’ in Ca2’-loaded muscles is mainly caused by greater Ca2+ release. The contraction induced by 0.1 mM Ca2’ was potentiated by a M$+ deficiency in the external medium. Because the tension development induced by 30 mM KC1 in normal PSS or by an introduction of 5 mM Ca2’ in OCa iso-K solution was not affected by the removal of external Mg+, the possible involvement of a change in Ca2+ influx or Ca2+ sensitivity of contractile apparatus caused by M$+ deficiency had a negligible influence on the augmented response to 0.1 mM Ca2’ in depolarized muscles. The potentiation similar to a potentiation of caffeine-induced contraction by Mg+-deficient solution (18, 30, 32) suggests that this was due to the enhanced Ca2’-induced Ca2’ release. The contraction due to 0.1 mM Ca2’ was also enhanced by lowering the temperature of the medium, whereas the contraction due to 5 mM Ca2+ was decreased by this procedure. This is also consistent with the observations that the low temperature enhances a contraction due to Ca2+-induced Ca2’ release (6, 23, 32). Furthermore, ryanodine exhibited use-dependent inhibition on the contraction, which means that opening Ca2+ release channels augments the action of ryanodine (2). These findings support the involvement of Ca2+-induced Ca2+ release in the Ca contraction. We can therefore conclude that the Ca2’ influx through voltage-dependent Ca2+ channels triggered the release of Ca2+ from the SR. The Ca2+ release by caffeine from SR of vascular smooth muscle is transient either in Ca2+-containing or Ca2+-free medium (4, 14, 32). This means that the SR cannot be reprimed with Ca2+ in the presence of caffeine even if Ca2+ is present in the cytoplasm. Ryanodine inhibited the entire course of Ca contraction over a 20min observation period when the added Ca2’ was 0.1 or
IN
VASCULAR
SMOOTH
MUSCLE
0.5 mM. Therefore, unlike the Ca2+ release by caffeine, the Ca2+ influx-induced Ca2+ release is likely to continuously occur as long as Ca2+ influx persists to refill the SR When 0.1 mM Ca2’ was applied to Ca2+-depleted muscle, the contraction at 2 min in the absence or presence of ryanodine was 10e5 M (20,24) or 2 X 10B6 M (5) in isolated skeletal or cardiac SR vesicles. In addition, the concentration of cytoplasmic Ca2+ required to inactivate the Ca2+-induced Ca2’ release mechanism in vascular smooth muscle has not been examined. Therefore it remains unclear whether the inactivation of the mechanism can occur with a physiological concentration of Ca2’ in intact vascular
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CA*+
INFLUX
AND CA*+ RELEASE
smooth muscles. Another possibility regarding the smaller ryanodinesensitive fraction when Ca2+ influx was high is that Ca2’ which entered cells through Ca2+ channels was sufficient to induce a maximum contraction by itself and surpassed the involvement of the Ca2+-induced Ca2’ release. As shown in Fig. 5, however, 0.5 and 1.0 mM Ca2’ caused the same magnitude of steady contraction, but the inhibition by ryanodine was larger with lower Ca2+. This suggests that Ca2’ -induced Ca2’ release plays a minor role in 1 mM Ca2’-induced contraction compared with that by 0.5 mM Ca2’ despite the same tension development. Himpens et al. (8) measured the intracellular Ca2’ signal by fi.ua-2 when Ca2+ was applied to depolarized rabbit pulmonary artery. The application of 0.65 mM Ca2’ caused an almost maximal contraction, as did 1 mM Ca2+, but the elevation of intracellular Ca2’ was slightly less with 0.65 mM Ca2+ than with 1 mM Ca2’. Similarly, in our study, the 45Ca2+ influx from extracellular fluid was less with 0.5 mM Ca2’ than with 1.0 mM Ca2+. Therefore the tension development of depolarized muscles due to >l mM Ca2+ may not be proportional to intracellular Ca2+ concentration. A further study with an intracellular Ca2+ indicator is needed to determine whether or how much Ca2’ release occurs when a high Ca2’ concentration is used. In any case the present data suggest that the inactivation of Ca2’ release by cytoplasmic Ca2+, if any, occurs at the Ca2+ concentration which by itself causes nearly maximal contraction with no help of Ca2+ release. In conclusion, this study showed that if Ca2’ influx via voltage-dependent Ca2+ channels is in the physiological range, Ca2’-induced Ca2’ release exerts a significant contribution to contractions. We thank Dr. J. L. Sutko for careful review of the manuscript. This work was partly supported by Ministry of Education, Science and Culture of Japan Grant 02660312. Address for reprint requests: K. Ito, Dept. of Veterinary Pharmacology, Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan. Received 5 November 1990; accepted in final form 17 June 1991.
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