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Intracellular site of “Ca reversal”: Inhibition of uterine smooth muscle contraction in Ca-free medium by a minute amount of Ca ion released from mitochondria by drugs
Gen. Pharmac. Vol. 17, No. 2, pp. 151 156, 1986 Printed in Great Britain. All rights reserved
0306-3623/86 $3.00+0.00 Copyright ~ 1986 Pergamon Press Ltd
I N T R A C E L L U L A R SITE OF "Ca REVERSAL": INHIBITION OF U T E R I N E S M O O T H M U S C L E C O N T R A C T I O N IN Ca-FREE M E D I U M BY A M I N U T E A M O U N T OF Ca ION R E L E A S E D F R O M M I T O C H O N D R I A BY D R U G S KIYOSHI SAKAI, TAKEHIRO YAMAGISHI and MASAATSU K. UCHIDA* Department of Molecular Pharmacology, Meiji College of Pharmacy, 1-35-23 Nozawa, Setagaya-ku, Tokyo 154, Japan (Received 17 May 1985)
Abstract--l. Sodium azide (NAN3) and dinitrophenol (DNP) at low concentrations caused "Ca reversal", i.e. inhibition of oxytocin-induced tonic contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution. 2. This inhibition was not due to depletion of ATP by NaN 3 or DNP. 3. Higher concentrations of NaN 3 and DNP caused additional contraction. 4. NaN a and DNP dose-dependently released Ca ion from mitochondria isolated from the estrogendominated rat uterine smooth muscle in vitro. 5. Electron microscopic studies have shown that in estrogen-dominated rat uterine smooth muscle cells, cytoplasmic membranes proliferate, resulting in compartmentalization of the myofilament-sarcoplasmic system and its separation from the receptor-effector system in the surface folds of the plasma membranes, which also contain some mitochondria. 6. It is proposed that low concentrations of NaN 3 and DNP release a small amount of Ca ion from these outer mitochondria and this Ca ion acts on the intracellular "site of Ca reversal" to induce reversal, i.e. inhibition. 7. Higher concentrations of NaN 3 and DNP are proposed to release a large amount of Ca ion from the central mitochondria near myofilaments and so induce contraction. 8. The "site of Ca reversal" was shown to be intracellular as our previous postulation.
INTRODUCTION
Previously, we reported that a minute amount of Ca ion inhibits contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution after prolonged incubation of the muscle with 3 m M E G T A . This inhibitory effect, which was named " C a reversal", may be related to stabilization of the cell in the resting state and threshold phenomena, because the EDs0-concentration calculated according to Imai and Takeda (1967) for this inhibition is ca. 4 x 10 -s M free Ca ion, which is no more than in the resting state (Sakai and Uchida, 1980; Sakai et al., 1981, 1982, 1983, 1984, 1985). U n d e r our conditions, this inhibitory effect was clearly manifested, but in ordinary conditions with a normal concentration o f Ca ion this effect cannot be observed. Since this inhibition by Ca ion of the contraction of the muscle was observed with various agonists (Sakai et al., 1985), we think this type of inhibition by a minute amount of Ca ion may be on the effector or on the post-receptor mechanisms. In a previous paper (Sakai et al., 1984), the following findings were reported: (a) Sustained contraction to oxytocin in Ca-free solution after prolonged preincubation with 3 m M E G T A (Sakai and Uchida, 1980; Sakai et al., 1981, 1982) was manifested only when the Ca-free medium contained E G T A to chelate Ca ion (free Ca ion; 10 -7 M). A low concentration of Ca ion of 10-SM contaminating the *To whom all correspondence should be addressed. 151
medium without E G T A did not have such an appreciable effect on the development of the maximal tension, but it had an adverse effect on maintenance of the tension; the decay of tension was observed. (b) Oxytocin-induced Ca-free contraction could be repeated with little reduction in magnitude only when the Ca-free medium contained E G T A . When it was contaminated with free Ca ion, i.e. in the absence of E G T A , progressive decrease in the magnitude of tension was observed in repeated trials and change in the pattern of contraction was observed. But the effect of omission of E G T A was reversible, as shown by reincubation of the tissue with 3 m M E G T A . These inhibitory effects of contaminating Ca ion are the manifestation of " C a reversal" phenomenon and the effects were not immediate. If the action sites of such a minute amount of Ca ion were superficial, the effect should be immediate. Thus the effect seems to be due to limited and progressive influx of contaminating Ca ion. Indeed, in the muscle chemically skinned, the inhibition was observed even at 10-8-10 -7 M Ca ion and the effect was immediate. So we postulated that the site of Ca reversal is intracellular. If this postulation is correct, Ca ion released intracellularly should cause this inhibition, i.e. Ca reversal. In the present work, we are going to confirm this postulation with drugs that release Ca ion from intracellular stores. Here, the released Ca ion should not induce contraction before it exerts inhibition, i.e. Ca reversal. Mitochondria had been thought to be a source of Ca ion for contraction. But Somlyo et al. (1979) showed by electron microprobe analysis that
152
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m i t o c h o n d r i a in s m o o t h muscle d o n o t show any change in Ca m o v e m e n t when the muscle contracts or relaxes. M o r e o v e r 3akai et al. (1978) showed by a n in vitro study with m i t o c h o n d r i a from estrogend o m i n a t e d rat uterine s m o o t h muscle t h a t mitochondrial C a u p t a k e is n o t affected by relaxants. Janis et al. (1977) reported t h a t in rat uterine s m o o t h muscle, microsomes or the sarcoplasmic reticulum has m u c h higher affinity for C a ion t h a n mitochondria. T h u s the sarcoplasmic reticulum, which is in close c o n t a c t with contractile proteins, takes u p Ca ion released from m i t o c h o n d r i a before it reaches the contractile elements so far as the released C a ion is n o t massive. R a e y m a e k e r s et al. (1977) also reported t h a t m i t o c h o n d r i a were n o t related with c o n t r a c t i o n o f guinea pig taenia caecum. So we tested drugs t h a t release C a ion from m i t o c h o n d r i a such as D N P a n d N a N 3 to determine w h e t h e r they inhibit c o n t r a c t i o n to oxytocin in Ca-free solution, t h a t is, w h e t h e r those drugs induce " C a reversal" o f muscle contraction.
MATERIALS AND METHODS Contraction o f rat uterine smooth muscle to oxytocin in Ca-free solution
Wistar rats, weighing 170-220g, were ovariectomized. After a recovery period of 5 days or more, the rats were given estradiol benzoate s.c. (0.1 mg/kg) once a day for 4 days and then killed. The uterine horns were removed and mounted in a carbogen-aerated bath with Locke-Ringer solution of the following composition (mM): NaCI 154, KCI 5.63, CaCI 2 2.16, MgCI 2 2.10, NaHCO 3 5.95 and glucose 5.55. Ca-free solution had the same composition except that CaCI 2 was omitted. The muscles were equilibrated in Locke-Ringer solution for 1 hr with a resting tension of 0.5 g and then incubated with 3 mM EGTA in Ca-free solution for 1 hr with a resting tension of 0.2 g. Then the solution was replaced by Ca-free solution containing 0.2 mM EGTA. Ten minutes after change of the medium, contraction was induced by addition of oxytocin (10 2 unit/ml). After the contraction reached a plateau, NaN~ (3 x 10-43 x 10 ~M) or dinitrophenol (DNP) (3 x 10-7-3 x 10 -3 M) was applied cumulatively. In some cases, the contraction was recorded
isolate uterine horns from estrogen-dominated rats (8-10 animals)
I wash with ice-cold Ca-free Locke-Ringer solution
I free from endometrium and blood vessels
I mince at 4°C with scissors
I homogenize in a Polytron PT-IO in 3 volumes of 0.32 M sucrose for three 5sec periods at a rheostat setting of 6
I rehomogenate in a Potter-homogenizer with a Teflon pestle in 10 volumes of 0.32 M sucrose 1200g for lOmin at 4°C I
I
precipitate
supernatant
I
I filter through two layers of gauze
discarded
15,000g for 15min at 4°C
I
I precipitate
supernatant
I 40,000 g for 60 min at 4°C
suspend in MS-buffer* 12,000 g for 15min at 4°C
precipitate
I precipitate
suspend in 0.6 M KCI-MOPS**
I 40,000 g for 60 min at 4°C
resuspend in KCI-buffer***
II [Mitochondrial fraction]
precipitate
I resuspend in KCI-buffer***
II [Microsomal fraction]
Fig. 1. Isolation of membrane fractions from rat uterine smooth muscle. *MS-buffer: mannitol 225 mM, sucrose 75 mM and MOPS 20 mM (pH 7.2 at 30°C). **0.6 M KCI-MOPS: KCl 0.6 M and MOPS 20 mM (pH 7.2 at 30°C). ***KCl-buffer: KCl 120 mM, MgC12 5 mM and MOPS 20 mM (pH 7.0 at 30'~C).
"Ca reversal" by intracellular Ca ion
153
~ 1 Oxytocin
1 NON3( M )
lO-2unit/ml
3x10-4
"[o.~ 1
1
20rain I
10-3
I
3 x 1 0 -3
Fig. 2. Ca reversal by NaN 3 of oxytocin-induced contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution after prolonged preincubation with 3 mM EGTA. NaN 3 caused relaxation at concentrations of 3 x 10 -4 M and 10 -3 M. However, 10 -3 M N a N 3 caused gradual increase in tension and 3 × 10 3 M NaN 3 caused only contraction.
after addition of NaN 3 or DNP (10 -4 M) and then Ca ion (10-4-3 × 10 -3 M) was added cumulatively to test whether NaN 3 or DNP inhibited contraction by depletion of energy sources. Preparation o f mitochondrial and microsomal fractions Mitochondrial and microsomal fractions were prepared as described before (Sakai et al., 1978) with a slight modification. Details of the procedure are shown in Fig. 1. Effects o f N a N 3 on 45Ca movements in mitochondrial and microsomal fractions The mitochondrial fraction was resuspended in medium containing 120mM KCI, 5mM MgC12, 20#M CaC12 (45Ca 30nCi/ml), 5mM glutamate and 20mM MOPS[3-(N-Morpholino)propane sulfonic acid] buffer (pH 7.0 at 30°C), the final protein concentration being 0.1 mg/ml. Protein was determined by Lowry's method (Lowry et al., 1951). For testing the effect of NAN3, the suspension was incubated for 15 min in the presence of ATP at 30°C to saturate mitochondrial Ca uptake and I min later various concentrations of NaN 3 were added and incubation was continued. At intervals a small amount of the suspension was filtered through millipore filters (HA, 0.45 #m) and the filters were washed with 2 ml of chilled reaction medium without Ca ion. Radioactivity on the filter was
counted in liquid scintillation counter in toluene based scintillator. The microsomal fraction was resuspended in medium containing 120 mM KCI, 5 mM MgCI 2, I # M CaCI2 (45Ca 30 nCi/ml), 5 mM oxalate and 20 mM MOPS buffer (pH 7.0 at 30°C). The reaction was started by adding 3 mM ATP and NaN 3 was added 1 min before ATP. At intervals, small amounts of the reaction mixture were filtered and counted as for the mitochondrial fraction. Statistical significance of difference was evaluated by Student's t-test and P = 0.05 was taken as the upper limit of significance. Agents used were DNP (2A-dinitrophenol) from Wako Pure Chemicals, Tokyo (for use usually dissolved in Ca-free solution, but in some cases dissolved in acetone as a stock solution, where the concentration of acetone had no effect on the muscle when added to make the final concentration of DNP 10-2 M in the bath) and NaN3 (Sodium azide) from Nakarai Chemicals, Kyoto (for use dissolved in water). Other agents were of analytical grade as described in the foregoing papers. RESULTS As s h o w n in Fig. 2, N a N 3 (3 x 1 0 - 4 - 1 0 - 3 M ) caused relaxation o f e s t r o g e n - d o m i n a t e d rat uterine
I
f Oxytocin
DNP ( M )
lO-Zunit/ml
10 -5
O.5g
3x 10 - 5
I0.5¢
f-f Oxy focin
DNP (M) 10- 6
10-2 unit/ml 3x 1 0 - '
10- 5 3x10 -6
3x 10-3
10- 3
3x10 -5
I 0.59 Oxytocin
DNP
Ca 2. (M)
10-2uni//ml
10-4M
10 - 4
5 x 10 - 4
10 - 3
5 x 10 - 3
20rain
Fig. 3. Ca reversal induced by dinitrophenol (DNP) of oxytocin-induced contraction of estrogendominated rat uterine smooth muscle in Ca-free solution after prolonged incubation with 3 mM EGTA. (a) DNP caused relaxation at concentrations of l0 -5 M and 3 x l0 -5 M dose-dependently. (b) DNP caused relaxation at concentrations up to 10 -3 M dose-dependently but contraction at 3 x l0 -3 M. (c) In the presence of l0 -4 M DNP, which was sufficient to cause relaxation, Ca ion at concentrations of 10-4-3 x l0 -3 M caused dose-dependent contraction. The magnitudes of the Ca-induced contraction were the same as in the absence of DNP (Sakai and Uchida, 1980). Dots (o) above trace (c) show values above scale.
154
K1YOSHISAKAIet al.
DISCUSSION 150"
¢U
o
100"
Q.
E o
E c
50 el
[]
• : NaN 3 ,3X10-4M
el ¢,J
Z~: NAN3,
10-3M
17: NaN3,3XlO-3M
* P<0.05 **
P
***P
15
1~
I
J
20
2/,
/
NaN 3
Time (rain)
Fig. 4. Release of Ca ion by NaN 3 from Ca-loaded mitochondria isolated from estrogen-dominated rat uterine smooth muscle layer. The mitochondria were incubated with 4~Ca, ATP and glutamate for 15 min, when the uptake of Ca ion reached a plateau. Then 1 min later NaN 3 was added and the Ca content of the mitochondria was measured at intervals by filtrating samples on millipore filters. NaN 3 dose-dependent release of Ca ion from mitochondria. smooth muscle contracted by oxytocin in Ca-free solution after prolonged incubation with 3 m M EGTA. The relaxation, named "Ca reversal" by Sakai and Uchida (1980), was dependent on the dose of NaN 3. Figure 3 shows that DNP had a similar effect. Figure 3(a) shows relaxation of the contraction by DNP (10-5-3 x 10 5 M). Figure 3(b) shows the dosedependency of the relaxation, i.e. Ca reversal, on the DNP concentration. Higher concentrations of DNP (3 x 10-3M) and NaN 3 (3 x 10 -3M) caused contraction of the once relaxed muscle by lower concentrations of the same agents (Figs 2 and 3(b)). In the presence of DNP (10 4M), which was enough to cause Ca reversal, exogenously applied Ca ion (3 x 10-4-3 x 10 -3 M) induced the same tension developments (Fig. 3(c)) as in the absence of DNP, as reported by Sakai and Uchida (1980). With NaN 3 (3 x 10 -4 M) the same result was obtained (data not shown). NaN 3 (3 x 10-4-3 x 10 -3 M) caused a dosedependent release of Ca ion from isolated mitochondria from estrogen-dominated rat uterine smooth muscle which were loaded with 45Ca ion in the presence of ATP and glutamate in vitro (Fig. 4). These results were the same as observed with DNP in mitochondria from various origins (Vashington and Murphy, 1962; Carafoli et al., 1975), which were confirmed by us on our preparation. NaN 3 and DNP had no effect on microsomal Ca ion movements.
NaN3 and DNP caused a dose-dependent reversal of oxytocin-induced contraction of estrogendominated rat uterine smooth muscle in Ca-free solution after prolonged incubation with 3mM EGTA. This inhibition was not caused by depletion of energy source, ATP, because in the presence of NaN3 and DNP at sufficient concentrations to induce Ca reversal, addition of Ca ion at concentrations that induced contraction caused the same tension as in the absence of DNP and NaN 3. In contrast to low concentrations of NaN 3 and DNP, higher concentrations of these compounds induced contraction. In in vitro studies, NaN 3 released Ca ion dosedependently from isolated mitochondria loaded with Ca ion and a similar effect of DNP was reported by Vashington and Murphy (1962), Vallieres et al. (1975) and Carafoli et al. (1975). Van Breemen et al. (1975) reported that metabolic inhibitors such as DNP stimulated Ca ion efflux from smooth muscle cells, indicating Ca ion release from the intracellular store, mitochondria. At low concentrations, NaN 3 and DNP caused release of a small amount of Ca ion and induced the reversal, while in higher concentrations they caused release of a large amount of the ion and contraction of the muscle. These agents can readily penetrate the cell. Thus it is concluded that the reversal was induced by Ca ion released intracellularly and that it can be identified as "Ca reversal". These findings well coincide with the previous postulation that the site of Ca reversal is intracellular. Ca reversal is the relaxation of contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution, which was preincubated with 3 mM EGTA. This contraction can be repeated with little reduction in magnitude when EGTA is present in the Ca-free medium (Sakai et al., 1984). Contraction, or strictly, actin-myosin interaction without Ca ion and its inhibition by Ca ion was observed in lower eukaryotes (Kohama and Kendrick-Jones, 1982; Tominaga et al., 1983; Kikuyama and Tazawa, 1983) but the contraction of the uterine smooth muscle of the rat in Ca-free solution seems to be triggered by Ca ion (Sakai et al., 1983). As there is no Ca ion outside the cell, the Ca ion for contraction must be supplied by its release from intracellular stores, and the absence of decay in contraction height in repeated contractions may be due to recycling of Ca ion between sarcoplasmic reticulum (SR) and myofilaments in the cell without its extrusion through the plasma membrane from the cell. This needs compartmentalization of the myofilament-SR system in the cell. Consistent with this idea, proliferation of cytoplasmic membrane has been observed in the estrogendominated rat uterine smooth muscle cells by Ross and Klebanoff (1967). They observed that on treatment of rat with estradiol benzoate after castration, uterine smooth muscle cells displayed extensive enlargement with dilatation of SR and Golgi complex. These cytoplasmic membranes were located close together not only in the perinuclear zone but also close to the cell surface in so-called "outpouchings" or folds of the cell surface. Myofilaments were present in the cytoplasm interspersed between the two
155
"Ca reversal" by intracellular Ca ion ~-" Site
•
.-t.~_
jr
"t,~," ..
l
of Ca Reversal
~
dh
Oxylocin receptor
~.~ . - ' t~ .
proliferotedl cytoplasmic membranes
.j ~
Effector
~
/
/ 'or.........
plasma membrane
\ |
/
,.,~¢ . "o ~ ... ?• , C" O ~
~,...{."
",
""
~.~_~.
myofiloments
.:, ", .t:F ~
'.
;.
,
..w,.--"
'~I.:h."
Fig. 5. Diagram of part of an estrogen-dominated rat uterine smooth muscle cell based on the electronmicroscopic observation of Ross and Klebanoff 0967). Compartmentalization by proliferated cytoplasmic membranes of the receptor-effector system and of the sarcoplasmic reticulum-myofilament system are shown. In the latter compartment located in the inner part of the cell, sustained contraction may be induced by Ca ion released from the sarcoplasmic reticulum, which may be directly caught by myofilaments (or calmodulin) without affecting the cytoplasmic concentration of Ca ion in the former compartment. Dinitrophenol and azide at low concentrations may act on the mitochondria in the former compartment located in the outer part of the cell or in the outpouchings to release minute amounts of Ca ion and induce Ca reversal. This diagram also shows our hypothetical "Site of Ca Reversal" and the actions of dinitrophenol (DNP) and azide (NAN3) and influx of Ca ion. layers of the proliferated cytoplasmic membranes (Fig. 5). The proliferated membranes close to the cell surface compartmentalize the myofilament-SR system and this may be why there was no decay of repeated contractions; the Ca ion released from SR should be immediately taken up by the contractile system nearby and vice versa without extrusion through plasma membrane. High doses of NaN 3 and DNP may cause contraction by releasing Ca ion from mitochondria in large amounts. These proliferated membranes may compartmentalize the receptor-effector system at the same time. There should be a very low concentration of Ca ion in the outpouchings. The outpouchings contain a few mitochondria and low doses of NaN 3 and DNP presumably first act on them to release a minute amount of Ca ion and cause Ca reversal (Fig. 5). Higher concentrations of the agents must cause contraction mainly by inducing massive release of Ca ion from mitochondria near the myofilaments. As shown in Fig. 2, l0 -3 M NaN3 first cause relaxation and then gradually contraction of the muscle. This can be explained by supposing that it first acted on mitochondria in the outpouchings to cause Ca reversal but took time to reach the mitochondria near the myofilaments. Thus "'the site of Ca reversal" seems to
be near the receptor-effector system, that is, in the outpouchings near the inner surface of the cell. These explanations are consistent with our findings that in EGTA-permeabilized tissues a minute amount of exogenous Ca ion (10-s-I 0-7 M) caused Ca reversal by its influx (Sakai et al., 1984). We confirmed that the "site of Ca reversal" is intracellular and seems to be located near the inner surface of the plasma membrane. Usually the inhibitory action is masked by excitatory activities of Ca ion per se but in this case it could be demonstrated because of the morphological changes induced by estrogen and because of the unique experimental conditions used. At present, it cannot be denied that this inhibition might be an artifact under nonphysiological experimental conditions but our working hypothesis is that such a minute amount of Ca ion may play stabilizing roles in the resting state of the cell, because the concentration of Ca ion to induce this inhibition is just its intracellular concentration in the resting state.
SUMMARY Sodium azide and dinitrophenol, which in vitro release Ca ion from mitochondria isolated from
KIYOSHI SAKAIet al.
156
estrogen-dominated rat uterine smooth muscle, caused Ca reversal (relaxation) of oxytocin-induced contraction of the muscle in Ca-free solution, which was preincubated with 3 m M E G T A . This inhibition was not due to depletion of energy source. Higher doses of azide and dinitrophenol induced contraction. Namely a minute, a m o u n t of Ca ion is released from mitochondria by a low dose of azide or dinitrophenol acts on the intracellular "site of Ca reversal" to induce Ca reversal. The present findings are consistent with previous findings that Ca reversal is induced by minute amount of intracellular Ca ion (ca. 10-8-10 -7 M). This inhibitory action of Ca ion may be manifested by the compartmentalization of the myofilaments-sarcoplasmic reticulum system by estrogen-domination.
REFERENCES
Carafoli E., Malmstr6m K., Capano M., Sigel E. and Crmpton M. (1975) Mitochondria and the regulation of cell calcium. In Calcium Transport in Contraction and Secretion (Edited by Carafoli F. and Drabikowski W.) pp. 53-64. North-Holland Publishing Co., Amsterdam. Imai S. and Takeda K. (1967) Effect ofvasodilator upon the isolated taenia coli of the guinea pig. J. Pharmac. exp. Ther. 156, 557-564. Janis R. A., Crakshaw D. J. and Daniel E. E. (1977) Control of intracellular Ca 2+ activity in rat myometrium. Am. J. Physiol. 232, C50-C58. Kikuyama M. and Tazawa M. (1983) Transient increase of intracellular-Ca 2+ during excitation of tonoplast-free Chara cells. Protoplasma 117, 62-67. Kohama K. and Kendrick-Jones J. (1982) Ca-dependent inhibitory factor for the myosin-actin-ATP interaction by Physarum polycephalum. J. Muscle Res. Cell Motility 3, 491-501. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent../, biol. Chem. 193, 265-275. Raeymaekers L., Wuytak F., Batra S. and Casteels R. (1977) A comparison study of the Calcium accumulation by mitochondria and microsomes isolated from the smooth muscle of the guinea pig taenia coli. Pfliigers Arch. Ges. Physiol. 368, 217-223. Ross R. and Klebanoff S. J. (1967) Fine structural changes
in uterine smooth muscle and fibroblasts in response to estrogen. J. Cell Biol. 32, 155 167. Sakai K. and Uchida M. (1980) A Calcium reversal phenomenon: Differentiation of excitatory and inhibitory roles of Ca in uterine smooth muscle contraction. Jap. J. Pharmac. 30, 394-396. Sakai K., Takayanagi I., Uchida M. and Takagi K. (1978) Effect of papaverine on calcium ion uptake by a mitochondrial fraction isolated from rat uterine smooth muscle. Eur. J. Pharmac. 50, 131 136. Sakai K. Yamaguchi T. and Uchida M. (1981) Oxytocininduced Ca-free contraction of rat uterine smooth muscle: Effects of divalent cations and drugs. Arch. int. Pharmacodyn. Ther. 250, 40-54. Sakai K., Higuchi K., Yamaguchi T. and Uchida M. (1982) Oxytocin-induced Ca-free contraction of rat uterine smooth muscle: Effects of preincubation with EGTA and drugs. Gen. Pharmac. 13, 393 399. Sakai K., Yamaguchi T., Morita S. and Uchida M. (1983) Agonist-induced contraction of rat myometrium in Cafree solution containing Mn. Gen. Pharmac. 14, 391-400. Sakai K., Yamaguchi T., Higuchi-Nakamura K. and Uchida M. K. (1984) Calcium reversal: Intracellular localization of site of inhibition by a submicromolar concentration of Ca ion in uterine smooth muscle shown by studies with EGTA. Gen. Pharmac. 15, 549-552. Sakai K., Higuchi-Nakamura K. and Uchida M. K. (1985) Ca reversal: Inhibition by Ca ion of sustained contraction in Ca-free medium induced by various agonists in rat uterine smooth muscle. Gen. Pharmac. 16, 133-136. Somlyo A. P., Somlyo A. V. and Shuman H. (1979) Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei and cytoplasm. J. Cell Biol. 81, 316-335. Tominaga Y., Shimmen T. and Tazawa M. (1983) Control of cytoplasmic streamings by extracellular Ca 2+ in permealized Nitella cells. Protoplasma 116, 75-77. Vallieres J., Scarpa A. and Somlyo A. P. (1975) Subcellular fractions of smooth muscle. Isolation, substrate utilization and Ca 2÷ transport by main pulmonary artery and mesenteric vein mitochondria. Arch. Biochem. Biophys. 170, 659-671. Van Breeman C. J., Wuytack F. and Casteels R. (1975) Stimulation of 45Ca efl~lux from smooth muscle cells by metabolic inhibition and high K depolarization. Pfliigers Arch. Ges. Physiol. 359, 183-196. Vashington F. D. and Murphy J. V. (1962) Ca 2÷ uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J. biol. Chem. 237, 2670-2677.
0306-3623/86 $3.00+0.00 Copyright ~ 1986 Pergamon Press Ltd
I N T R A C E L L U L A R SITE OF "Ca REVERSAL": INHIBITION OF U T E R I N E S M O O T H M U S C L E C O N T R A C T I O N IN Ca-FREE M E D I U M BY A M I N U T E A M O U N T OF Ca ION R E L E A S E D F R O M M I T O C H O N D R I A BY D R U G S KIYOSHI SAKAI, TAKEHIRO YAMAGISHI and MASAATSU K. UCHIDA* Department of Molecular Pharmacology, Meiji College of Pharmacy, 1-35-23 Nozawa, Setagaya-ku, Tokyo 154, Japan (Received 17 May 1985)
Abstract--l. Sodium azide (NAN3) and dinitrophenol (DNP) at low concentrations caused "Ca reversal", i.e. inhibition of oxytocin-induced tonic contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution. 2. This inhibition was not due to depletion of ATP by NaN 3 or DNP. 3. Higher concentrations of NaN 3 and DNP caused additional contraction. 4. NaN a and DNP dose-dependently released Ca ion from mitochondria isolated from the estrogendominated rat uterine smooth muscle in vitro. 5. Electron microscopic studies have shown that in estrogen-dominated rat uterine smooth muscle cells, cytoplasmic membranes proliferate, resulting in compartmentalization of the myofilament-sarcoplasmic system and its separation from the receptor-effector system in the surface folds of the plasma membranes, which also contain some mitochondria. 6. It is proposed that low concentrations of NaN 3 and DNP release a small amount of Ca ion from these outer mitochondria and this Ca ion acts on the intracellular "site of Ca reversal" to induce reversal, i.e. inhibition. 7. Higher concentrations of NaN 3 and DNP are proposed to release a large amount of Ca ion from the central mitochondria near myofilaments and so induce contraction. 8. The "site of Ca reversal" was shown to be intracellular as our previous postulation.
INTRODUCTION
Previously, we reported that a minute amount of Ca ion inhibits contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution after prolonged incubation of the muscle with 3 m M E G T A . This inhibitory effect, which was named " C a reversal", may be related to stabilization of the cell in the resting state and threshold phenomena, because the EDs0-concentration calculated according to Imai and Takeda (1967) for this inhibition is ca. 4 x 10 -s M free Ca ion, which is no more than in the resting state (Sakai and Uchida, 1980; Sakai et al., 1981, 1982, 1983, 1984, 1985). U n d e r our conditions, this inhibitory effect was clearly manifested, but in ordinary conditions with a normal concentration o f Ca ion this effect cannot be observed. Since this inhibition by Ca ion of the contraction of the muscle was observed with various agonists (Sakai et al., 1985), we think this type of inhibition by a minute amount of Ca ion may be on the effector or on the post-receptor mechanisms. In a previous paper (Sakai et al., 1984), the following findings were reported: (a) Sustained contraction to oxytocin in Ca-free solution after prolonged preincubation with 3 m M E G T A (Sakai and Uchida, 1980; Sakai et al., 1981, 1982) was manifested only when the Ca-free medium contained E G T A to chelate Ca ion (free Ca ion; 10 -7 M). A low concentration of Ca ion of 10-SM contaminating the *To whom all correspondence should be addressed. 151
medium without E G T A did not have such an appreciable effect on the development of the maximal tension, but it had an adverse effect on maintenance of the tension; the decay of tension was observed. (b) Oxytocin-induced Ca-free contraction could be repeated with little reduction in magnitude only when the Ca-free medium contained E G T A . When it was contaminated with free Ca ion, i.e. in the absence of E G T A , progressive decrease in the magnitude of tension was observed in repeated trials and change in the pattern of contraction was observed. But the effect of omission of E G T A was reversible, as shown by reincubation of the tissue with 3 m M E G T A . These inhibitory effects of contaminating Ca ion are the manifestation of " C a reversal" phenomenon and the effects were not immediate. If the action sites of such a minute amount of Ca ion were superficial, the effect should be immediate. Thus the effect seems to be due to limited and progressive influx of contaminating Ca ion. Indeed, in the muscle chemically skinned, the inhibition was observed even at 10-8-10 -7 M Ca ion and the effect was immediate. So we postulated that the site of Ca reversal is intracellular. If this postulation is correct, Ca ion released intracellularly should cause this inhibition, i.e. Ca reversal. In the present work, we are going to confirm this postulation with drugs that release Ca ion from intracellular stores. Here, the released Ca ion should not induce contraction before it exerts inhibition, i.e. Ca reversal. Mitochondria had been thought to be a source of Ca ion for contraction. But Somlyo et al. (1979) showed by electron microprobe analysis that
152
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m i t o c h o n d r i a in s m o o t h muscle d o n o t show any change in Ca m o v e m e n t when the muscle contracts or relaxes. M o r e o v e r 3akai et al. (1978) showed by a n in vitro study with m i t o c h o n d r i a from estrogend o m i n a t e d rat uterine s m o o t h muscle t h a t mitochondrial C a u p t a k e is n o t affected by relaxants. Janis et al. (1977) reported t h a t in rat uterine s m o o t h muscle, microsomes or the sarcoplasmic reticulum has m u c h higher affinity for C a ion t h a n mitochondria. T h u s the sarcoplasmic reticulum, which is in close c o n t a c t with contractile proteins, takes u p Ca ion released from m i t o c h o n d r i a before it reaches the contractile elements so far as the released C a ion is n o t massive. R a e y m a e k e r s et al. (1977) also reported t h a t m i t o c h o n d r i a were n o t related with c o n t r a c t i o n o f guinea pig taenia caecum. So we tested drugs t h a t release C a ion from m i t o c h o n d r i a such as D N P a n d N a N 3 to determine w h e t h e r they inhibit c o n t r a c t i o n to oxytocin in Ca-free solution, t h a t is, w h e t h e r those drugs induce " C a reversal" o f muscle contraction.
MATERIALS AND METHODS Contraction o f rat uterine smooth muscle to oxytocin in Ca-free solution
Wistar rats, weighing 170-220g, were ovariectomized. After a recovery period of 5 days or more, the rats were given estradiol benzoate s.c. (0.1 mg/kg) once a day for 4 days and then killed. The uterine horns were removed and mounted in a carbogen-aerated bath with Locke-Ringer solution of the following composition (mM): NaCI 154, KCI 5.63, CaCI 2 2.16, MgCI 2 2.10, NaHCO 3 5.95 and glucose 5.55. Ca-free solution had the same composition except that CaCI 2 was omitted. The muscles were equilibrated in Locke-Ringer solution for 1 hr with a resting tension of 0.5 g and then incubated with 3 mM EGTA in Ca-free solution for 1 hr with a resting tension of 0.2 g. Then the solution was replaced by Ca-free solution containing 0.2 mM EGTA. Ten minutes after change of the medium, contraction was induced by addition of oxytocin (10 2 unit/ml). After the contraction reached a plateau, NaN~ (3 x 10-43 x 10 ~M) or dinitrophenol (DNP) (3 x 10-7-3 x 10 -3 M) was applied cumulatively. In some cases, the contraction was recorded
isolate uterine horns from estrogen-dominated rats (8-10 animals)
I wash with ice-cold Ca-free Locke-Ringer solution
I free from endometrium and blood vessels
I mince at 4°C with scissors
I homogenize in a Polytron PT-IO in 3 volumes of 0.32 M sucrose for three 5sec periods at a rheostat setting of 6
I rehomogenate in a Potter-homogenizer with a Teflon pestle in 10 volumes of 0.32 M sucrose 1200g for lOmin at 4°C I
I
precipitate
supernatant
I
I filter through two layers of gauze
discarded
15,000g for 15min at 4°C
I
I precipitate
supernatant
I 40,000 g for 60 min at 4°C
suspend in MS-buffer* 12,000 g for 15min at 4°C
precipitate
I precipitate
suspend in 0.6 M KCI-MOPS**
I 40,000 g for 60 min at 4°C
resuspend in KCI-buffer***
II [Mitochondrial fraction]
precipitate
I resuspend in KCI-buffer***
II [Microsomal fraction]
Fig. 1. Isolation of membrane fractions from rat uterine smooth muscle. *MS-buffer: mannitol 225 mM, sucrose 75 mM and MOPS 20 mM (pH 7.2 at 30°C). **0.6 M KCI-MOPS: KCl 0.6 M and MOPS 20 mM (pH 7.2 at 30°C). ***KCl-buffer: KCl 120 mM, MgC12 5 mM and MOPS 20 mM (pH 7.0 at 30'~C).
"Ca reversal" by intracellular Ca ion
153
~ 1 Oxytocin
1 NON3( M )
lO-2unit/ml
3x10-4
"[o.~ 1
1
20rain I
10-3
I
3 x 1 0 -3
Fig. 2. Ca reversal by NaN 3 of oxytocin-induced contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution after prolonged preincubation with 3 mM EGTA. NaN 3 caused relaxation at concentrations of 3 x 10 -4 M and 10 -3 M. However, 10 -3 M N a N 3 caused gradual increase in tension and 3 × 10 3 M NaN 3 caused only contraction.
after addition of NaN 3 or DNP (10 -4 M) and then Ca ion (10-4-3 × 10 -3 M) was added cumulatively to test whether NaN 3 or DNP inhibited contraction by depletion of energy sources. Preparation o f mitochondrial and microsomal fractions Mitochondrial and microsomal fractions were prepared as described before (Sakai et al., 1978) with a slight modification. Details of the procedure are shown in Fig. 1. Effects o f N a N 3 on 45Ca movements in mitochondrial and microsomal fractions The mitochondrial fraction was resuspended in medium containing 120mM KCI, 5mM MgC12, 20#M CaC12 (45Ca 30nCi/ml), 5mM glutamate and 20mM MOPS[3-(N-Morpholino)propane sulfonic acid] buffer (pH 7.0 at 30°C), the final protein concentration being 0.1 mg/ml. Protein was determined by Lowry's method (Lowry et al., 1951). For testing the effect of NAN3, the suspension was incubated for 15 min in the presence of ATP at 30°C to saturate mitochondrial Ca uptake and I min later various concentrations of NaN 3 were added and incubation was continued. At intervals a small amount of the suspension was filtered through millipore filters (HA, 0.45 #m) and the filters were washed with 2 ml of chilled reaction medium without Ca ion. Radioactivity on the filter was
counted in liquid scintillation counter in toluene based scintillator. The microsomal fraction was resuspended in medium containing 120 mM KCI, 5 mM MgCI 2, I # M CaCI2 (45Ca 30 nCi/ml), 5 mM oxalate and 20 mM MOPS buffer (pH 7.0 at 30°C). The reaction was started by adding 3 mM ATP and NaN 3 was added 1 min before ATP. At intervals, small amounts of the reaction mixture were filtered and counted as for the mitochondrial fraction. Statistical significance of difference was evaluated by Student's t-test and P = 0.05 was taken as the upper limit of significance. Agents used were DNP (2A-dinitrophenol) from Wako Pure Chemicals, Tokyo (for use usually dissolved in Ca-free solution, but in some cases dissolved in acetone as a stock solution, where the concentration of acetone had no effect on the muscle when added to make the final concentration of DNP 10-2 M in the bath) and NaN3 (Sodium azide) from Nakarai Chemicals, Kyoto (for use dissolved in water). Other agents were of analytical grade as described in the foregoing papers. RESULTS As s h o w n in Fig. 2, N a N 3 (3 x 1 0 - 4 - 1 0 - 3 M ) caused relaxation o f e s t r o g e n - d o m i n a t e d rat uterine
I
f Oxytocin
DNP ( M )
lO-Zunit/ml
10 -5
O.5g
3x 10 - 5
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f-f Oxy focin
DNP (M) 10- 6
10-2 unit/ml 3x 1 0 - '
10- 5 3x10 -6
3x 10-3
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I 0.59 Oxytocin
DNP
Ca 2. (M)
10-2uni//ml
10-4M
10 - 4
5 x 10 - 4
10 - 3
5 x 10 - 3
20rain
Fig. 3. Ca reversal induced by dinitrophenol (DNP) of oxytocin-induced contraction of estrogendominated rat uterine smooth muscle in Ca-free solution after prolonged incubation with 3 mM EGTA. (a) DNP caused relaxation at concentrations of l0 -5 M and 3 x l0 -5 M dose-dependently. (b) DNP caused relaxation at concentrations up to 10 -3 M dose-dependently but contraction at 3 x l0 -3 M. (c) In the presence of l0 -4 M DNP, which was sufficient to cause relaxation, Ca ion at concentrations of 10-4-3 x l0 -3 M caused dose-dependent contraction. The magnitudes of the Ca-induced contraction were the same as in the absence of DNP (Sakai and Uchida, 1980). Dots (o) above trace (c) show values above scale.
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K1YOSHISAKAIet al.
DISCUSSION 150"
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Fig. 4. Release of Ca ion by NaN 3 from Ca-loaded mitochondria isolated from estrogen-dominated rat uterine smooth muscle layer. The mitochondria were incubated with 4~Ca, ATP and glutamate for 15 min, when the uptake of Ca ion reached a plateau. Then 1 min later NaN 3 was added and the Ca content of the mitochondria was measured at intervals by filtrating samples on millipore filters. NaN 3 dose-dependent release of Ca ion from mitochondria. smooth muscle contracted by oxytocin in Ca-free solution after prolonged incubation with 3 m M EGTA. The relaxation, named "Ca reversal" by Sakai and Uchida (1980), was dependent on the dose of NaN 3. Figure 3 shows that DNP had a similar effect. Figure 3(a) shows relaxation of the contraction by DNP (10-5-3 x 10 5 M). Figure 3(b) shows the dosedependency of the relaxation, i.e. Ca reversal, on the DNP concentration. Higher concentrations of DNP (3 x 10-3M) and NaN 3 (3 x 10 -3M) caused contraction of the once relaxed muscle by lower concentrations of the same agents (Figs 2 and 3(b)). In the presence of DNP (10 4M), which was enough to cause Ca reversal, exogenously applied Ca ion (3 x 10-4-3 x 10 -3 M) induced the same tension developments (Fig. 3(c)) as in the absence of DNP, as reported by Sakai and Uchida (1980). With NaN 3 (3 x 10 -4 M) the same result was obtained (data not shown). NaN 3 (3 x 10-4-3 x 10 -3 M) caused a dosedependent release of Ca ion from isolated mitochondria from estrogen-dominated rat uterine smooth muscle which were loaded with 45Ca ion in the presence of ATP and glutamate in vitro (Fig. 4). These results were the same as observed with DNP in mitochondria from various origins (Vashington and Murphy, 1962; Carafoli et al., 1975), which were confirmed by us on our preparation. NaN 3 and DNP had no effect on microsomal Ca ion movements.
NaN3 and DNP caused a dose-dependent reversal of oxytocin-induced contraction of estrogendominated rat uterine smooth muscle in Ca-free solution after prolonged incubation with 3mM EGTA. This inhibition was not caused by depletion of energy source, ATP, because in the presence of NaN3 and DNP at sufficient concentrations to induce Ca reversal, addition of Ca ion at concentrations that induced contraction caused the same tension as in the absence of DNP and NaN 3. In contrast to low concentrations of NaN 3 and DNP, higher concentrations of these compounds induced contraction. In in vitro studies, NaN 3 released Ca ion dosedependently from isolated mitochondria loaded with Ca ion and a similar effect of DNP was reported by Vashington and Murphy (1962), Vallieres et al. (1975) and Carafoli et al. (1975). Van Breemen et al. (1975) reported that metabolic inhibitors such as DNP stimulated Ca ion efflux from smooth muscle cells, indicating Ca ion release from the intracellular store, mitochondria. At low concentrations, NaN 3 and DNP caused release of a small amount of Ca ion and induced the reversal, while in higher concentrations they caused release of a large amount of the ion and contraction of the muscle. These agents can readily penetrate the cell. Thus it is concluded that the reversal was induced by Ca ion released intracellularly and that it can be identified as "Ca reversal". These findings well coincide with the previous postulation that the site of Ca reversal is intracellular. Ca reversal is the relaxation of contraction of estrogen-dominated rat uterine smooth muscle in Ca-free solution, which was preincubated with 3 mM EGTA. This contraction can be repeated with little reduction in magnitude when EGTA is present in the Ca-free medium (Sakai et al., 1984). Contraction, or strictly, actin-myosin interaction without Ca ion and its inhibition by Ca ion was observed in lower eukaryotes (Kohama and Kendrick-Jones, 1982; Tominaga et al., 1983; Kikuyama and Tazawa, 1983) but the contraction of the uterine smooth muscle of the rat in Ca-free solution seems to be triggered by Ca ion (Sakai et al., 1983). As there is no Ca ion outside the cell, the Ca ion for contraction must be supplied by its release from intracellular stores, and the absence of decay in contraction height in repeated contractions may be due to recycling of Ca ion between sarcoplasmic reticulum (SR) and myofilaments in the cell without its extrusion through the plasma membrane from the cell. This needs compartmentalization of the myofilament-SR system in the cell. Consistent with this idea, proliferation of cytoplasmic membrane has been observed in the estrogendominated rat uterine smooth muscle cells by Ross and Klebanoff (1967). They observed that on treatment of rat with estradiol benzoate after castration, uterine smooth muscle cells displayed extensive enlargement with dilatation of SR and Golgi complex. These cytoplasmic membranes were located close together not only in the perinuclear zone but also close to the cell surface in so-called "outpouchings" or folds of the cell surface. Myofilaments were present in the cytoplasm interspersed between the two
155
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Fig. 5. Diagram of part of an estrogen-dominated rat uterine smooth muscle cell based on the electronmicroscopic observation of Ross and Klebanoff 0967). Compartmentalization by proliferated cytoplasmic membranes of the receptor-effector system and of the sarcoplasmic reticulum-myofilament system are shown. In the latter compartment located in the inner part of the cell, sustained contraction may be induced by Ca ion released from the sarcoplasmic reticulum, which may be directly caught by myofilaments (or calmodulin) without affecting the cytoplasmic concentration of Ca ion in the former compartment. Dinitrophenol and azide at low concentrations may act on the mitochondria in the former compartment located in the outer part of the cell or in the outpouchings to release minute amounts of Ca ion and induce Ca reversal. This diagram also shows our hypothetical "Site of Ca Reversal" and the actions of dinitrophenol (DNP) and azide (NAN3) and influx of Ca ion. layers of the proliferated cytoplasmic membranes (Fig. 5). The proliferated membranes close to the cell surface compartmentalize the myofilament-SR system and this may be why there was no decay of repeated contractions; the Ca ion released from SR should be immediately taken up by the contractile system nearby and vice versa without extrusion through plasma membrane. High doses of NaN 3 and DNP may cause contraction by releasing Ca ion from mitochondria in large amounts. These proliferated membranes may compartmentalize the receptor-effector system at the same time. There should be a very low concentration of Ca ion in the outpouchings. The outpouchings contain a few mitochondria and low doses of NaN 3 and DNP presumably first act on them to release a minute amount of Ca ion and cause Ca reversal (Fig. 5). Higher concentrations of the agents must cause contraction mainly by inducing massive release of Ca ion from mitochondria near the myofilaments. As shown in Fig. 2, l0 -3 M NaN3 first cause relaxation and then gradually contraction of the muscle. This can be explained by supposing that it first acted on mitochondria in the outpouchings to cause Ca reversal but took time to reach the mitochondria near the myofilaments. Thus "'the site of Ca reversal" seems to
be near the receptor-effector system, that is, in the outpouchings near the inner surface of the cell. These explanations are consistent with our findings that in EGTA-permeabilized tissues a minute amount of exogenous Ca ion (10-s-I 0-7 M) caused Ca reversal by its influx (Sakai et al., 1984). We confirmed that the "site of Ca reversal" is intracellular and seems to be located near the inner surface of the plasma membrane. Usually the inhibitory action is masked by excitatory activities of Ca ion per se but in this case it could be demonstrated because of the morphological changes induced by estrogen and because of the unique experimental conditions used. At present, it cannot be denied that this inhibition might be an artifact under nonphysiological experimental conditions but our working hypothesis is that such a minute amount of Ca ion may play stabilizing roles in the resting state of the cell, because the concentration of Ca ion to induce this inhibition is just its intracellular concentration in the resting state.
SUMMARY Sodium azide and dinitrophenol, which in vitro release Ca ion from mitochondria isolated from
KIYOSHI SAKAIet al.
156
estrogen-dominated rat uterine smooth muscle, caused Ca reversal (relaxation) of oxytocin-induced contraction of the muscle in Ca-free solution, which was preincubated with 3 m M E G T A . This inhibition was not due to depletion of energy source. Higher doses of azide and dinitrophenol induced contraction. Namely a minute, a m o u n t of Ca ion is released from mitochondria by a low dose of azide or dinitrophenol acts on the intracellular "site of Ca reversal" to induce Ca reversal. The present findings are consistent with previous findings that Ca reversal is induced by minute amount of intracellular Ca ion (ca. 10-8-10 -7 M). This inhibitory action of Ca ion may be manifested by the compartmentalization of the myofilaments-sarcoplasmic reticulum system by estrogen-domination.
REFERENCES
Carafoli E., Malmstr6m K., Capano M., Sigel E. and Crmpton M. (1975) Mitochondria and the regulation of cell calcium. In Calcium Transport in Contraction and Secretion (Edited by Carafoli F. and Drabikowski W.) pp. 53-64. North-Holland Publishing Co., Amsterdam. Imai S. and Takeda K. (1967) Effect ofvasodilator upon the isolated taenia coli of the guinea pig. J. Pharmac. exp. Ther. 156, 557-564. Janis R. A., Crakshaw D. J. and Daniel E. E. (1977) Control of intracellular Ca 2+ activity in rat myometrium. Am. J. Physiol. 232, C50-C58. Kikuyama M. and Tazawa M. (1983) Transient increase of intracellular-Ca 2+ during excitation of tonoplast-free Chara cells. Protoplasma 117, 62-67. Kohama K. and Kendrick-Jones J. (1982) Ca-dependent inhibitory factor for the myosin-actin-ATP interaction by Physarum polycephalum. J. Muscle Res. Cell Motility 3, 491-501. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent../, biol. Chem. 193, 265-275. Raeymaekers L., Wuytak F., Batra S. and Casteels R. (1977) A comparison study of the Calcium accumulation by mitochondria and microsomes isolated from the smooth muscle of the guinea pig taenia coli. Pfliigers Arch. Ges. Physiol. 368, 217-223. Ross R. and Klebanoff S. J. (1967) Fine structural changes
in uterine smooth muscle and fibroblasts in response to estrogen. J. Cell Biol. 32, 155 167. Sakai K. and Uchida M. (1980) A Calcium reversal phenomenon: Differentiation of excitatory and inhibitory roles of Ca in uterine smooth muscle contraction. Jap. J. Pharmac. 30, 394-396. Sakai K., Takayanagi I., Uchida M. and Takagi K. (1978) Effect of papaverine on calcium ion uptake by a mitochondrial fraction isolated from rat uterine smooth muscle. Eur. J. Pharmac. 50, 131 136. Sakai K. Yamaguchi T. and Uchida M. (1981) Oxytocininduced Ca-free contraction of rat uterine smooth muscle: Effects of divalent cations and drugs. Arch. int. Pharmacodyn. Ther. 250, 40-54. Sakai K., Higuchi K., Yamaguchi T. and Uchida M. (1982) Oxytocin-induced Ca-free contraction of rat uterine smooth muscle: Effects of preincubation with EGTA and drugs. Gen. Pharmac. 13, 393 399. Sakai K., Yamaguchi T., Morita S. and Uchida M. (1983) Agonist-induced contraction of rat myometrium in Cafree solution containing Mn. Gen. Pharmac. 14, 391-400. Sakai K., Yamaguchi T., Higuchi-Nakamura K. and Uchida M. K. (1984) Calcium reversal: Intracellular localization of site of inhibition by a submicromolar concentration of Ca ion in uterine smooth muscle shown by studies with EGTA. Gen. Pharmac. 15, 549-552. Sakai K., Higuchi-Nakamura K. and Uchida M. K. (1985) Ca reversal: Inhibition by Ca ion of sustained contraction in Ca-free medium induced by various agonists in rat uterine smooth muscle. Gen. Pharmac. 16, 133-136. Somlyo A. P., Somlyo A. V. and Shuman H. (1979) Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei and cytoplasm. J. Cell Biol. 81, 316-335. Tominaga Y., Shimmen T. and Tazawa M. (1983) Control of cytoplasmic streamings by extracellular Ca 2+ in permealized Nitella cells. Protoplasma 116, 75-77. Vallieres J., Scarpa A. and Somlyo A. P. (1975) Subcellular fractions of smooth muscle. Isolation, substrate utilization and Ca 2÷ transport by main pulmonary artery and mesenteric vein mitochondria. Arch. Biochem. Biophys. 170, 659-671. Van Breeman C. J., Wuytack F. and Casteels R. (1975) Stimulation of 45Ca efl~lux from smooth muscle cells by metabolic inhibition and high K depolarization. Pfliigers Arch. Ges. Physiol. 359, 183-196. Vashington F. D. and Murphy J. V. (1962) Ca 2÷ uptake by rat kidney mitochondria and its dependence on respiration and phosphorylation. J. biol. Chem. 237, 2670-2677.