Actapharmacol. et toxicol. 1978, 42, 185-193
From the Department of Pharmacology, Linkoping University, Regionsjukhusef S-581 85 Linkoping, Sweden
Some Properties of Ca-Binding Microsomal Subfractions Isolated from Rabbit Colon Muscle BY Karin B. Nilsson, Rolf G. G. Anderson, Ella Mohme-Lundholm and Lennart Lundholm (Received June 28, 1977; Accepted September 19, 1977)
Abstract: From a homogenate of rabbit colon smooth muscle a microsomal fraction was isolated, which was divided into subfractions by centrifugation on a discontinuous sucrose gradient. The Ca-binding properties of the subfractions were investigated under different conditions. In the presence of 0.35 mM ATP the Ca binding of the fractions amounted to 4-8 nmol/mg protein. The 35% fraction bound more Ca per mg protein than the 3545% fraction. The Ca accumulation was comparatively higher both in the presence of 5 mM ATP and in the presence of 5 mM oxalate. The two fractions showed about the same sensitivity for oxalate. This substance stimulated the Ca uptake at 5 mM but not at lower concentrations. The amount and the rate of Ca binding were more dependent on variations in the exogenous ATP concentration in the 35% fraction than was the case for the 3545% fraction. The Ca binding was completely inhibited by salyrgan when the microsomal fractions were pretreated with this agent. Sodium azide did not influence the Ca-binding capacity of the fractions. It is suggested that the microsomal subfractions of the rabbit colon muscle represent physiologically important parts of the Ca sequestering system of the muscle, since Ca binding takes place at Ca- and ATP-concentrations which are believed to be present in the myoplasm.
Key-words: Calcium - smooth muscle microsomes - rabbits.
Calcium ions possess several important regulatory functions in different kinds of tissues. In smooth muscle the role of Ca in the contraction-relaxation cycle has attracted much interest (Bianchi 1968; Hurwitz & Suria 1971; Ruegg 1971). By using histochemical techniques, Ca has been observed to be localized to different structures of the smooth muscle cell such as the plasma membrane, the invaginations of the plasma membrane (the “surface vesicles”), the sarcoplasmic reticulum, the mitochondria and the nucleus (Devine et al. 1973; Jonas & Zelck 1974; Popescu et al. 1974; Heumann 1976). During the last decade several investigators have isolated microsomal and mitochondria1 fractions from homogenates of different kinds of smooth muscle, which bind Ca particularly in the presence of MgATP. Subcellular fractions have been iso-
lated from the intestinal (Anderson & Nilsson 1972; Hurwitz et al. 1975; Is0 1975; Tomiyama et al. 1975; Rayemaekers et al. 1976), the vascular (Fitzpatrick et al. 1972; Baudouin-Legros & Meyer 1973; Hess & Ford 1974; Shibata & Hollander 1974; Zelck et al. 1974; Wei et al. 1976) and the uterine smooth muscle (Carsten 1969; Batra & Daniel 1971a; Jarris et al. 1977; Krall et al. 1976). Differences belween the Ca-binding properties of the microsomal and the mitochondrial fractions have been observed by several investigators (Carsten 1969; Batra 1972; Hess & Ford 1974). The crude microsomes of smooth muscle could be separated into subfractions by centrifugation on a sucrose density gradient (Carsten 1969; Anderson & Nilsson 1972; Hurwitz et al. 1973; Janis et al. 1977). In rabbit colon smooth muscle
186
CA-BINDING O F SMOOTH MUSCLE MICROSOMES
one subfraction (the 3545% fraction) contained structures from which Ca was released by cholinergic drugs and to which the Ca binding was increased by stimulation of P-adrenoceptors. The Ca release from or binding to the subfraction isolated at 35% sucrose was only to a minor extent influenced by cholinergic or adrenergic drugs (Nilsson & Anderson 1977; Nilsson er al. 1977). The aim of this investigation was to study some basal Ca-binding properties of the two subfractions. Materials and Methods Preparation of microsomal fractions. Microsomal fractions from rabbit colon were isolated according to Carsten (1969) with some modifications (Nilsson et al. 1978). The mitochondrial fraction was isolated at 17,300x g for 20 min. The crude microsomal fraction obtained after centrifugation at 40,000 x g for 90 min. was suspended in 0.08 M-NaCI and 0.005 M-Na,-oxalate (pH 7.0). The suspension was placed on a sucrose density gradient and centrifuged for 2 hrs at 50,000 x g. Three protein layers were obtained, a narrow band in the upper part of the 35% sucrose, “the 35% fraction”, a wide protein layer in the 35% and 45% sucrose layers and a narrow band at 45% and 55% sucrose. The two former fractions were used in this investigation as the yield of protein was very low in the third fraction (Nilsson et al. 1978). The fractions were stored at 4” for about 20 hrs until used for the Ca-accumulation studies. The mitochondrial fraction and the crude microsomes were in some Ca-binding experiments suspended in 40% sucrose. Determination of Ca accumulation. The accumulation of Ca to the subcellular fractions was studied at 37” in 1 ml of a standard incubation medium containing 0. I 1 M-KC1, 0.01 M-NaCI, 0.02 M histidine buffer (pH 7.2). 0.35 mM-MgCl,, 0.35 mM ATP, CaC1, and 0.008 pci ““a. The total calcium concentration including impurities of the chemicals was M by atomic absorption spectroestimated to 9 x photometry. In some experiments the Ca binding was investigated in the presence of oxalate and/or at different ATP concentrations as indicated in the legends. The reaction was started by the addition of about lo& 150 Fg protein. After different time periods the reaction was interrupted by filtration through Millipore filters (0.45 pm). Aliquots of the filtrate were dissolved in 10 ml InstagelO solution and the radioactivity of “ T a was counted in a Packard Tri Carb liquid scintillation spectrometer. The amount of 45Ca bound to the different fractions was calculated from the differences in
the radioactivity between the unfiltered and the filtered reaction mixtures. Correction was made for 45Ca absorbed to the filter. The free Ca concentration in the incubation solution was determined according to Katz et al. (1970) using EGTA (ethyleneglycol-bis(p-aminoethylether)-N, ”-tetraacetic acid) and by varying the amount of total Ca as indicated in table 1. When the Ca binding of the 3545% fraction was determined by the Millipore filtration technique and by analyzing the absolute increase in Ca by atomic absorption spectrophotometry, similar values were obtained, which is in accordance with other investigators (Batra & Daniel 1971b; Baudouin-Legros & Meyer 1973). Chemical assays. The ATP and glucose -6-phosphate content of the microsomes was determined according to Lamprecht & Trautschold (1970). The protein concentration of the fractions was determined according to Lowry et al. (1951) using serum albumin as standard. Statistical analysis. Mean values are given f standard error of the mean. The samples in each separate experiment were obtained from the same microsomal preparation. The significance was calculated by Student’s t-test from the difference between the control and the treated samples.
Results The Ca-binding capacity of a crude microsomal fraction isolated in the range of 17,300 x g for 20 min. -+ 40,000 x g for 90 min. is shown in fig. 1. There was a rapid binding of Ca for the first 2 min. after which the binding increased more slowly. Some accumulation of Ca still occurred after 10 min. incubation.
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Fig. 1. The Ca binding of the crude microsomal fraction. The data are the average of six different experiments S.E.M.
187
KARIN B. NILSSON ET AL.
The Ca binding of microsomal subfractions under standard conditions.
The crude microsomes were separated into subfractions by centrifugation on a discontinuous sucrose gradient. The rate of the Ca binding at 37" was very rapid during the first 2-3 min. in both the 35% and the 3 5 4 5 % fractions, whereas during the following minutes there was only a minor increase (fig. 2). The Ca-binding capacity of the 3 5 4 5 % fraction after 10 min. incubation and was amounted to 4 . 7 k 1.5 nmol/mg_protein .
about half of that estimated in the 35% fraction, 8.5 0.9 nmol/mg protein. The Ca binding at different free CaZ+-concentrations.
It was of interest to investigate the C a binding of the microsomal fractions in relation to the free CaZ -concentration of the incubation solution. These experiments were performed at pH =6.8. As indicated in table 1 no Ca2+ was bound at 1 x lo-* M but there was a limited binding at 1.9~ M . The binding was increased with rising Ca2 concentrations up to a free Ca2 concentration of 5.8 x M. The microsomes were thus able to bind Ca at the Ca concentrations thought to be present in the myoplasm of smooth muscle. +
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Fig. 2. The effects of ATP and temperature on the Ca binding of the 35% fraction (a) and of the 3 5 4 5 % fraction (b). The values represent the mean of 6 determinations S,E,M in the presence of o,35 mM ATP at 370;.--. in the presence of 0.35 m~ ATP at 4 ; .- x - x ~. in the absence of ATP at 37". ~
+
The ej'ect of oxalate on the Caz' -uptake. To skeletal muscle microsomes, added Caprecipitating anions, such as oxalate, increased the Ca uptake by precipitating Ca oxalate into the microsomal vesicles when the solubility product was reached (Hasselbach & Makinose 1963; Weber et al. 1966). The effect of oxalate on the rabbit colon microsomalCa uptake under standard conditions was studied in the concentration range of 0 to 5 mM. The Ca uptake of the 35% and 3 5 4 5 % fractions was stimulated at the highest concentration ( 5 mM) but not at the lower ones (fig. 3). The increase was evident after 5 min. At this point 5 m M oxalate increased the uptake of the 35% fraction by about 20% compared to the value obtained in the absence of oxalate (fig. 3a) and in the 3 5 4 5 % fraction the corresponding increase was about 90% (fig. 3b). In an incubation medium containing 5 m M MgATP and 5 m M oxalate the C a uptake of the 35% and 3 5 4 5 % fractions was initially rather slow, but after 3 min. rapidly increased. After this time it was almost linear with time up to a t least 20 min. (fig. 4), but still no equilibrium of the Ca uptake was reached. In the presence of 0.35 mM ATP and in the absence of oxalate the binding was maximal within 10 min. After 20 min. the Ca accumulation of the 35% and 3545% fractions in the presence of 5 mM MgATP
188
KARIN B. NILSSON ET AL. Table 1
The effect of the free Ca2+concentrations on the Ca binding of the microsomal subfractions. The microsomal fractions were incubated for 5 min. in solutions containing varying concentrations of ionized Caz+calculated according to Katz et al. (1970) by combining EGTA (ethyleneglycol-bis-(P-amhoethylether)-N,N-tetraacetic acid) with the total Ca at pH=6.8. The Ca added was measured with an atomic absorption spectrophotometer. Mean k S.E.M. n=6. Caz+ (MI
Total calcium (MI
EGTA (M)
1.ox 10-8
3.7x 1 0 - 5 3 . 6 1~0 - 5 4.0~ 9.0 x
0.57 x lo-, 0 . 5 9 ~10-3 0.79 x
1.2 x 1 0 - 7 1.9~ 5.8 x
~
and 5 mM oxalate was 5-7-fold that of the binding reached at 0.35 mM MgATP. Temperature.
The rate and the amount of Ca bound to the submicrosomal fractions was influenced by changes in the temperature. When the temperature of the standard incubation solution was reduced from 37" to 4" the amount of Ca bound by the 3 5 4 5 % fraction was markedly reduced (fig. 2b). There was no significant binding for the first 5 min. but after 10 min. some binding occurred. In the 35% fraction a binding of Ca occurred even during the first minute at 4" and this accumulation changed very little during the following 10 min. (fig. 2a).
Ca (nmol/mg protein) 35% 35-45% 0.0 0.013 k0.008 0.59 k0.29 3.90 k0.67
0.0 0.022 k 0.010 0.43 k0.18 3.98 k0.60
somewhat higher, 55.6f 3.9 nmol/mg protein. The hexosephosphate might thus be used for the regeneration of ATP. The time-response curve of the Ca binding of the 35% fraction indicated that the binding was a
b
The influence of ATP on the Ca binding.
The binding of Ca to the microsomal fractions was an energy dependent reaction. In the absence of exogenous ATP there was some binding of Ca to the 35% fraction after 1 min. but the binding did not increase with the time (fig. 2a). The 3545% fraction also bound some Ca in absence of exogenous ATP (fig. 2b). The question which arose was whether the binding was really independent of ATP or whether the microsomes eventually contained endogenous ATP and/or an ATP-generating system. Analysis of the microsoma1 fractions showed the presence of endogenous ATP in a concentration range of 4&70 nmol/mg protein. The glucose-6-phosphate (G6-P) content of the 35% fraction was 33.4& 1.7 nmol/mg protein. In the 3 5 4 5 % fraction it was
1 rnin
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Fig. 3. The effect of different concentrations of Na,oxalate (CL5 mM) on the Ca uptake in the 35% fraction (a) and in the 3 5 4 5 % fraction (b) after 1 and 5 min. of incubation. Before the reaction was started by the addition of microsomal proteins (lo&] 50 pg) Na,oxalte (pH=7.2) was added to the test tube. The data represent the mean values of five to six different experiments S.E.M. The statistical significance of the differences between the paired samples in the presence and in the absence of Na,-oxalate is denoted by *= P~0.05.
*
189
CA-BINDING OF SMOOTH MUSCLE MICROSOMES
initially lower in the presence of 5 m M ATP than in the presence of 0.35 mM ATP (fig. 5a). After 10 min. no equilibrium was reached at the high ATP concentration. To determine the ATP optimum of the binding reaction in the absence of oxalate a short incubation time was chosen. The
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relationship between the Ca binding and the ATP concentrations (0-1.05 mM) was then investigated. A maximum of the Ca binding of the 35% fraction was observed at 0.05 m M ATP (fig. 5b). The 3545% fraction showed less variation in Ca-binding capacity a t different ATP concentra-
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Fig. 4. Ca binding and Ca uptake in the 35% fraction (a) and in the 3545% fraction (b). The values are the S.E.M. in average of six different experiments the presence of 0.35 mM MgATP and without Na,oxalate, in the presence of 5 mM-MgATP and 5 mM-Na,-oxalate.
+
0.2
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0.6
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Fig. 5. a) The Ca binding of the 35% fraction in the presence of 0.35 mM-ATP (.--.) and in the presence of 5 mM ATP (.--.). The data represent the mean of S.E.M. b) The effect of ATP 6 different experiments (0-1.5 mM) on the Ca binding in the 35% fraction (.--.) and in the 3545% fraction (.--.). Before the incubation of the samples was started by addition of microsomal proteins different concentrations of MgATP (adjusted to pH=7.2) were added to the incubation medium. The fractions were incubated for two min. and three min. respectively. The data represent the mean of five to six separate determinations S.E.M.
+
190
KARIN B. NILSSON ET AL. Table 2
Influence of salyrganand sodium azide o n the Ca binding of the microsomal subfractions. 1) = Salyrgan and sodium azide were added simultaneously with the incubation medium containing MgATP. 2) = The microsomal proteins were pretreated for 5 min. by salyrgan before addition of the incubation medium. Mean k S.E.M. n=2-6. Ca binding (nmol/mg protein/lO min.)
Microsomes
35%
3545%
Control 3 mM salyrgan” 3 mM salyrgan’’ 5 mM-NaN,”
6.0k0.8 2.8k0.6
4.9k0.7 1.8 k0.6
0 6.4+ 1.0
0
3.9k0.5
tions. A maximal response seemed to be obtained at about 0.35 m M ATP under our experimental conditions (fig. 5b). Since very low concentrations of ATP stimulated the C a binding, the possibility had to be considered that endogenous ATP present in the microsomes participitated in the binding in the absence of exogenous ATP. However, this question needs further investigation. Calculated from the intact rabbit colon preparation the concentration of ATP in the water phase of the muscle was estimated to be about 0.3-1.5 m M (Anderson & Mohme-Lundholm 1970), a value which decided us to choose 0.35 mM ATP in the standard incubation medium.
Effect of salyrgan and sodium azide on the Ca binding. Salyrgan (3 mM), an inhibitor of microsomal Ca accumulation, reduced the Ca binding of the 35% and 3 5 4 5 % fractions t o 52% and 60% of the control values respectively, when the drug was added simultaneously with ATP. When the microsomal proteins were pretreated for 5 min. by salyrgan before the addition of the incubation medium containing ATP, the Ca binding was completely inhibited (table 2). The addition of 5 mM sodium azide, a n inhibitor of mitochondrial Ca accumulation did not influence the C a binding of the 35% or 3545% fractions (table 2).
Ca binding of the mitochondria1 fraction. It has not been the aim of this study to investigate the Ca-binding properties of the mitochondrial fraction. It was found, however, that the 3 5 4 5 % subfraction contained some activity of cytochrome c oxidase and whole as well as damaged mitochondria were observed on the electron micrographs (Nilsson et al. 1977b). For purposes of comparison the Ca binding of the mitochondrial fraction was studied under the same experimental conditions, with the same incubation medium used in the investigations on the microsomal subfractions. The C a binding of the mitochondrial fraction was only observed in the fresh preparation and was lost rapidly during storage (fig. 6). After storage for 24 hrs at 4” no C a binding was detectable. Sodium azide (5 mM), an inhibitor of mitochondrial Ca accumulation, reduced the Ca binding of the fresh mitochondrial fraction by about 40%. Since the C a binding of the microsomal fractions was always investigated after cold storage for 20-24 hrs the contribution of mitochondria or mitochondrial fragments t o the C a binding of the 3 5 4 5 % fraction was probably insignificant under our experimental conditions.
Discussion Fig. 6 . The effect of storage (C24 hrs) on the Ca binding of the mitochondrial fraction. The Ca binding was studied in the same incubation medium as used for microsomal subfractions.
In this study it is shown that the crude microsomal fraction of the rabbit colon muscle can be resolved into three subfractions. Our results indicate
CA-BINDING OF SMOOTH MUSCLE MICROSOMES
that the microsomal subfractions isolated from rabbit colon smooth muscle differed somewhat regarding their Ca-binding capacity. The Cabinding per mg protein of the 35% fraction was usually greater than that of the 3 5 4 5 % fraction, when investigated under our standard conditions. There were also indications that the Ca binding of the 35% fractions was more influenced by the variation in the ATP concentration than that of the 3 5 4 5 % fraction (fig. 5). Beta-adrenergic agonists increased the Ca binding and cholinergic agonists released Ca from the 3 5 4 5 % fraction, whereas these drugs had no or very weak effects on the 35% fraction (Anderson et al. 1975; Nilsson & Anderson 1977; Nilsson et al. 1977). Differences were also observed in the activities of some enzymes as well as in the morphological pictures of the two fractions. These observations will be discussed in a subsequent paper (Nilsson et al. 1978). When combining these findings the 350/, fraction seemed to consist mainly of vesicles, which may have their origin from the plasma membrane as well as from the sarcoplasmic reticulum. The 3 5 4 5 % fraction probably consisted of fragments of the sarcolemma and some mitochondria1 fragments. The mitochondria were probably not involved in the Ca binding of the 3 5 4 5 % fraction under our experimental conditions, as the mitochondria completely lost their Ca-binding capacity after storage for 24 hrs at 4" (fig. 6). We also observed that sodium azide did not inhibit Ca binding of the 3 5 4 5 % fraction or in the 35% fraction (table 2). There are other observations which indicate the presence of microsomes with different Ca-binding properties in smooth muscle. Thus from vascular smooth muscle some investigators have isolated oxalate-sensitive microsomes (Fitzpatrick et al. 1972; Hess & Ford 1974; Shibata & Hollander 1974; Ford & Hess 1975; Webb & Bhalla 1976) while others found the microsomes to be oxalateinsensitive (Allen 1973; Baudouin-Legros & Meyer 1973; Zelck et al. 1975). Zelcket al. (1975), applying identical experimental techniques in studies on microsomes from pig coronary artery and from guinea pig ileum, found that the first mentioned microsomes were oxalate-sensitive whereas the others were not.
191
Our results show that the MgATP concentration is of importance for demonstrating convincing effects of oxalate. At high MgATP concentrations oxalate stimulated the Ca uptake about 3fold in the two microsomal subfractions (fig. 3,4), while only minor effects were observed at the lower MgATP concentrations (fig. 3). The Ca uptake in colon microsomes was stimulated by 5 m M oxalate; this concentration was also needed for stimulating the Ca uptake in vascular smooth muscle microsomes (Ford & Hess 1975). The relationship between Ca binding and ATPconcentration wascomplex. Most research workers (Batra & Daniel 1971a; Fitzpatrick et al. 1972; Baudouin-Legros & Meyer 1973; Hess & Ford 1974; Shibata & Hollander 1974; Zelck et al. 1974; Tomiyama et al. 1975; Wei et al. 1976) have investigated the Ca accumulation of smooth muscle microsomes at 3-5 m M ATP, which is about 8-fold the mean concentration of the intact intestinal and vascular smooth muscle (Anderson 1973 ; Anderson & Mohme-Lundholm 1970). The time course of the Ca accumulation of the 35% microsomes was not equal when studied at 0.35 and 5 mM ATP (fig. 5a). At the lower concentration most Ca binding took place within 2-3 min. whereas at the higher concentration the Ca uptake was initially rather slow but then increased gradually, but no equilibrium was reached within 10 min. The latter type of timecourse has been observed by those investigators who have used high ATP concentrations (Batra 1972; Fitzpatrick et al. 1972; Hurwitz et al. 1973; Hess & Ford 1974; Tomiyama er al. 1975). The time-course of the Ca accumulation in the presence of 0.35 m M ATP might better reflect the changes in cytoplasmic Ca concentration of colon muscle during relaxation than the more artificial medium containing 5 mM ATP and oxalate. Most of the binding of Ca occurred within the first minute, whereas in the intact colon muscle, for instance, P-adrenoceptor agonists require 30-60 sec. for complete relaxation (Anderson & MohmeLundholm 1970). The processes that lead to Ca binding in smooth muscles are still incompletely investigated. The cyclic AMP-protein kinase system has been suggested to be of importance for the P-adrenoceptor-
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KARIN B. NILSSON ET AL.
stimulated Ca binding t o microsomal fractions (Andersson et al. 1976a). In other studies (Andersson el al. 1976b) the ATP-dependent Ca accumulation of colon microsomes have been studied by the so-called ESCA-technique (Electron Spectroscopy f o r chemical analysis). By this technique it was shown that phosphorous (probably in phosphate-form) was incorporated into the microsomal membranes along with calcium, when the microsomes were in a medium containing MgATP. Thus one possible mechanism for microsomal Ca binding might be phosphorylation of microsomal membranes. Another part of the ATP-dependent Ca accumulation in colon microsomes which was stimulated by oxalate may be similar to the “Ca pump” observed in sarcoplasmic reticulum isolated from skeletal muscle (Weber 1966). Acknowledgements Financial support was provided by the Swedish State Medical Research Council (B76- 14X-0208010A, B76-04X-04498-02, B76-04X-00101-12A), the Magnus Bergvall Foundation a n d the Swedish Society of Medical Research. We a r e indebted t o Mrs. L. Mackerlova and M r . S. Borjesson for valuable technical assistance.
Allen, J. C. : Characteristics of a vesicular Ca’ binding fraction from canine aorta: aortic relaxing factor (ARF). Biophys. J . 1973,13, 103a. Andersson, R.: Role of cyclic AMP and C a + + in mechanical and metabolic events in isometrically contracting vascular smooth muscle. Acta physiol. scand. 1973,87, 84-95. Andersson, R. G. G., L. Djarv, K. Nilsson & J. Wikberg: The role of calcium and cyclic nucleotides in the regulation of tension in smooth muscle of the gastrointestinal tract. In : Stimulus-secretion coupling in the gastrointestinal tract. Eds.: Case & Goebell, Medical Technical Publ. Co., Lancaster, 1976a, pp. 1-15. Anderson, R. & E. Mohme-Lundholm: Metabolic actions in intestinal smooth muscle associated with relaxation mediated by adrenergic a- and P-receptors. Actaphysiol. scand. 1970, 79, 244-261. Anderson, R. & K. Nilsson : Cyclic AMP and calcium in relaxation in intestinal smooth muscle. Nature New Bid. 1972,238, 119-120. Andersson, R. G. G., K. B. Nilsson, K.-E. Magnusson & L. Johansson : Relationship between calcium accumulating structures and the cyclic AMP system in +
intestinal smooth muscle. Abstr. Fifh Znt. Congr. Histochemistry and Cytochemistry, Bucharest, 1976b, pp. 22. Andersson, R., K. Nilsson, J. Wikberg, S. Johansson, E. Mohme-Lundholm & L. Lundholm: Cyclic nucleotides and the contraction of smooth muscle. In: Advances in cyclic nucleoride research. Eds.: G. I. Drummond, P. Greengard & G . A. Robison, Raven Press, New York, 1975,5,491-518. Batra, S . C. : The role of calcium binding by subcellular particles in the contraction and relaxation of the myometrium. Am. J . Obstet. Gynicol. 1972, 112, 851-856. . Batra, S. C. & E. E. Daniel: ATP-dependent Ca uptake by subcellular fractions of uterine smooth muscle. Comp. Biochem. Physiol. 1971a, 38A, 369-385. Batra, S. C. & E. E. Daniel: Effect of multivalent cations and drugs on Ca uptake by the rat myometrial microsomes. Comp. Biochem. Physiol. 1971b, %A, 285-300. Baudouin-Legros, M. & P. Meyer: Effects of angiotensin, catecholamines and cyclic AMP on calcium transfer in aortic microsomes. Brit. J. Pharmacol. 1973, 47, 377-385. Bianchi, C. P. : Cellcalcium. Butterworths, London 1968, pp. 1-131. Carsten, M. E. : Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle. J. Gen. Physiol. 1969, 53, 414-426. Devine, C. A,, A. V. Somlyo & A. P. Somlyo: Sarcoplasmic reticulum and mitochondria as cation accumulation sites in smooth muscle. Phil. Trans. R . SOC.Lond. B. 1973,265, 17-23. Fitzpatrick, D. F., E. J. Landon, G. Debbas & L. Hurwitz: A calcium pump in vascular smooth muscle. Science 1972, 176, 305-306. Ford, G. D. & M. L. Hess: Calcium-accumulating properties of subcellular fractions of bovine vascular smooth muscle. Circ. Res. 1975, 37, 580-587. Hasselbach, W. & M. Makinose: Uber den Mechanismus des Calciumtransportes durch die Membranen des sarcoplasmatischen Reticulus. Biochem. Z . 1963, 339, 94-111. Hess, M. L. & G. D. Ford: Calcium accumulation by subcellular fractions from vascular smooth muscle. J . Mol. Cell. Cardiol. 1974,6, 275-282. Heumann, H.-G. : The subcellular localization of calcium in vertebrate smooth muscle: Calcium-containing and calcium-accumulating structures in muscle cells of mouse-intestine. Cell. Tiss. Res. 1976, 169, 22 1-23 1. Hurwitz, L., G. Debbas & S. Little: Effects of temperature and inorganic ions on calcium accumulation in microsomes from intestinal smooth muscle. Mol. Cell. Biochem. 1975,8, 31-41, Hurwitz, L., D. F. Fitzpatrick, G. Debbas & E. J. Landon: Localization of calcium pump activity in smooth muscle. Science, 1973, 179, 384-386.
CA-BINDING OF SMOOTH MUSCLE MICROSOMES Hurwitz, L. & A. Suria: The link between agonist action and response in smooth muscle. Ann. Rev. Pharmacol. 1971, 11, 327-350. Iso, T . : ATP-induced changes in microsomal Mg2' levels and relationships to muscle contraction in isolated guinea-pig ileum. Biochem. Pharmacol. 1975, 24, 2099-21 00. Janis, R. A,, D. J . Crankshaw & E. E. Daniel: Control of intracellular Ca2+ activity in rat myometrium. Amer. J . Physiol. 1977, 232, C50-C57. Jonas, L. & U. Zelck: The subcellular calcium distribution in the smooth muscle cells of the pig coronary artery. Exp. Cell Res. 1974, 89, 352-358. Katz, A. M., D. 1. Repke, J. E. Upshaw & M. A. Polascik : Characterization of dog cardiac microsomes. Use of zonal centrifugation to fractionate fragmented sarcoplasmicreticulum, (Na+ + K +)-activatedATPase and mitochondrial fragments. Biochim. Biophys. Act0 1970,205,473490. Krall, J . F., J. L. Swensen& S. G . Korenman: Hormonal control of uterine contraction characterization of cyclic AMP-dependent membrane properties in the myometrium. Biochim. Biophys. Acta 1976,448, 578588. Lamprecht, W. & I. Trautschold: Bestimmungen mit Hexokinase und Glucose-6-phosphat Dehydrogenase. In: Methoden der enzymatischen Analyse. Ed.: H. U . Bergmeyer. Verlag Chemie, Weinheim, 1970, pp. 20242033. Lowry, 0. H., N. J. Rosebrough, A. L. Farr & R. J. Randall: Protein measurement with the fohn phenal reagent. J . Biol. Chem. 1951, 193, 265-275. Nilsson, K. B. & R. G. G. Andersson: Effects of cholinergic drugs and imidazole on Ca release and cyclic AMP formation in microsomal fractions from rabbit colon. Acta pharmaeol. et to.xic01. 1977, 40, 389400. Nilsson, K. B., R. G. G . Andersson, S. Enestrom, L. Mackerlova & E. Mohme-Lundholm : Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle. Acta pharmacol. et toxicol. 1978. Nilsson, K. B., R. G . G . Andersson, E. Mohme-Lundholm & L. Lundholm: Cyclic AMP and Ca-binding
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in microsomal fractions isolated from rabbit colon smooth muscle. Acta pharmacol. et toxicol. 1977, 41, 53-64. Popescu, L. M . , 1. Diculescu, U. Zelck & N. lonescu: Ultrastructural distribution of calcium in smooth muscle cells of guinea-pig taenia coli. Cell Tiss. Res. 1974, 154. 357-378. Raeymaekers, L., F. Wuytack & R. Casteels: Calcium accumulation by mitochondria1 and microsomal fractions isolated from the smooth muscle of guinea-pig taenia coli. Arch. int. physiol. bioehem. 1976, 84, 355-357. Riiegg, J. C.: Smooth muscle tone. Physiol. Rev. 1971, 51, 201-248. Shibata, N. & W. Hollander: Studies on the role of arterial microsomes in the contractile function of the arteries. Exp. Mol. Pathol. 1974, 21, 1-15. Tomiyama, A,, I. Takanyagi & K. Takagi: Relationships between Ca2+ uptake by a microsomal fraction of guinea-pig taenia caecum and its relaxation. Biochem. Pharmacol. 1975, 24,9-12. Webb, R. C. & R. C. Bhalla: Calcium sequesteration by subcellular fractions isolated from vascular smooth muscle :Effect of cyclic nucleotides and prostaglandins. J . Mol. Cell Cardiol. 1976, 8, 145-157. Weber, A. : Energized calcium transport and relaxing factors. In: Current topics in bioenergetics. Ed.: Sonadi. Academic Press, New York, 1966, Vol. 1, pp. 203-254. Weber, A,, R. Herz & 1. Reiss: Study of the kinetics of calcium transport by isolated fragmented sarcoplasmic reticulum. Biochem. Z . 1966,345, 329-369. Wei, J.-W., R. A. Janis & E. E. Daniel: Isolation and characterization of plasma membrane from rat mesenteric arteries. Blood Vessels 1976, 13, 279-292. Zelck, U., L. Kenya & E. Albrecht: ATP-dependent calcium uptake by microsomal fractions of pig coronary artery and its dependence on bradykinin and angiotensin 11. Acta biol. med. germ. 1974, 32, KlLK5. Zelck, U., U. Karnstedt & E. Albrecht: Calcium uptake and calcium release by subcellular fractions of smooth muscle. Acta bid. med. germ. 1975, 34, 981-986.
From the Department of Pharmacology, Linkoping University, Regionsjukhusef S-581 85 Linkoping, Sweden
Some Properties of Ca-Binding Microsomal Subfractions Isolated from Rabbit Colon Muscle BY Karin B. Nilsson, Rolf G. G. Anderson, Ella Mohme-Lundholm and Lennart Lundholm (Received June 28, 1977; Accepted September 19, 1977)
Abstract: From a homogenate of rabbit colon smooth muscle a microsomal fraction was isolated, which was divided into subfractions by centrifugation on a discontinuous sucrose gradient. The Ca-binding properties of the subfractions were investigated under different conditions. In the presence of 0.35 mM ATP the Ca binding of the fractions amounted to 4-8 nmol/mg protein. The 35% fraction bound more Ca per mg protein than the 3545% fraction. The Ca accumulation was comparatively higher both in the presence of 5 mM ATP and in the presence of 5 mM oxalate. The two fractions showed about the same sensitivity for oxalate. This substance stimulated the Ca uptake at 5 mM but not at lower concentrations. The amount and the rate of Ca binding were more dependent on variations in the exogenous ATP concentration in the 35% fraction than was the case for the 3545% fraction. The Ca binding was completely inhibited by salyrgan when the microsomal fractions were pretreated with this agent. Sodium azide did not influence the Ca-binding capacity of the fractions. It is suggested that the microsomal subfractions of the rabbit colon muscle represent physiologically important parts of the Ca sequestering system of the muscle, since Ca binding takes place at Ca- and ATP-concentrations which are believed to be present in the myoplasm.
Key-words: Calcium - smooth muscle microsomes - rabbits.
Calcium ions possess several important regulatory functions in different kinds of tissues. In smooth muscle the role of Ca in the contraction-relaxation cycle has attracted much interest (Bianchi 1968; Hurwitz & Suria 1971; Ruegg 1971). By using histochemical techniques, Ca has been observed to be localized to different structures of the smooth muscle cell such as the plasma membrane, the invaginations of the plasma membrane (the “surface vesicles”), the sarcoplasmic reticulum, the mitochondria and the nucleus (Devine et al. 1973; Jonas & Zelck 1974; Popescu et al. 1974; Heumann 1976). During the last decade several investigators have isolated microsomal and mitochondria1 fractions from homogenates of different kinds of smooth muscle, which bind Ca particularly in the presence of MgATP. Subcellular fractions have been iso-
lated from the intestinal (Anderson & Nilsson 1972; Hurwitz et al. 1975; Is0 1975; Tomiyama et al. 1975; Rayemaekers et al. 1976), the vascular (Fitzpatrick et al. 1972; Baudouin-Legros & Meyer 1973; Hess & Ford 1974; Shibata & Hollander 1974; Zelck et al. 1974; Wei et al. 1976) and the uterine smooth muscle (Carsten 1969; Batra & Daniel 1971a; Jarris et al. 1977; Krall et al. 1976). Differences belween the Ca-binding properties of the microsomal and the mitochondrial fractions have been observed by several investigators (Carsten 1969; Batra 1972; Hess & Ford 1974). The crude microsomes of smooth muscle could be separated into subfractions by centrifugation on a sucrose density gradient (Carsten 1969; Anderson & Nilsson 1972; Hurwitz et al. 1973; Janis et al. 1977). In rabbit colon smooth muscle
186
CA-BINDING O F SMOOTH MUSCLE MICROSOMES
one subfraction (the 3545% fraction) contained structures from which Ca was released by cholinergic drugs and to which the Ca binding was increased by stimulation of P-adrenoceptors. The Ca release from or binding to the subfraction isolated at 35% sucrose was only to a minor extent influenced by cholinergic or adrenergic drugs (Nilsson & Anderson 1977; Nilsson er al. 1977). The aim of this investigation was to study some basal Ca-binding properties of the two subfractions. Materials and Methods Preparation of microsomal fractions. Microsomal fractions from rabbit colon were isolated according to Carsten (1969) with some modifications (Nilsson et al. 1978). The mitochondrial fraction was isolated at 17,300x g for 20 min. The crude microsomal fraction obtained after centrifugation at 40,000 x g for 90 min. was suspended in 0.08 M-NaCI and 0.005 M-Na,-oxalate (pH 7.0). The suspension was placed on a sucrose density gradient and centrifuged for 2 hrs at 50,000 x g. Three protein layers were obtained, a narrow band in the upper part of the 35% sucrose, “the 35% fraction”, a wide protein layer in the 35% and 45% sucrose layers and a narrow band at 45% and 55% sucrose. The two former fractions were used in this investigation as the yield of protein was very low in the third fraction (Nilsson et al. 1978). The fractions were stored at 4” for about 20 hrs until used for the Ca-accumulation studies. The mitochondrial fraction and the crude microsomes were in some Ca-binding experiments suspended in 40% sucrose. Determination of Ca accumulation. The accumulation of Ca to the subcellular fractions was studied at 37” in 1 ml of a standard incubation medium containing 0. I 1 M-KC1, 0.01 M-NaCI, 0.02 M histidine buffer (pH 7.2). 0.35 mM-MgCl,, 0.35 mM ATP, CaC1, and 0.008 pci ““a. The total calcium concentration including impurities of the chemicals was M by atomic absorption spectroestimated to 9 x photometry. In some experiments the Ca binding was investigated in the presence of oxalate and/or at different ATP concentrations as indicated in the legends. The reaction was started by the addition of about lo& 150 Fg protein. After different time periods the reaction was interrupted by filtration through Millipore filters (0.45 pm). Aliquots of the filtrate were dissolved in 10 ml InstagelO solution and the radioactivity of “ T a was counted in a Packard Tri Carb liquid scintillation spectrometer. The amount of 45Ca bound to the different fractions was calculated from the differences in
the radioactivity between the unfiltered and the filtered reaction mixtures. Correction was made for 45Ca absorbed to the filter. The free Ca concentration in the incubation solution was determined according to Katz et al. (1970) using EGTA (ethyleneglycol-bis(p-aminoethylether)-N, ”-tetraacetic acid) and by varying the amount of total Ca as indicated in table 1. When the Ca binding of the 3545% fraction was determined by the Millipore filtration technique and by analyzing the absolute increase in Ca by atomic absorption spectrophotometry, similar values were obtained, which is in accordance with other investigators (Batra & Daniel 1971b; Baudouin-Legros & Meyer 1973). Chemical assays. The ATP and glucose -6-phosphate content of the microsomes was determined according to Lamprecht & Trautschold (1970). The protein concentration of the fractions was determined according to Lowry et al. (1951) using serum albumin as standard. Statistical analysis. Mean values are given f standard error of the mean. The samples in each separate experiment were obtained from the same microsomal preparation. The significance was calculated by Student’s t-test from the difference between the control and the treated samples.
Results The Ca-binding capacity of a crude microsomal fraction isolated in the range of 17,300 x g for 20 min. -+ 40,000 x g for 90 min. is shown in fig. 1. There was a rapid binding of Ca for the first 2 min. after which the binding increased more slowly. Some accumulation of Ca still occurred after 10 min. incubation.
i
i
6
8
10 min
Fig. 1. The Ca binding of the crude microsomal fraction. The data are the average of six different experiments S.E.M.
187
KARIN B. NILSSON ET AL.
The Ca binding of microsomal subfractions under standard conditions.
The crude microsomes were separated into subfractions by centrifugation on a discontinuous sucrose gradient. The rate of the Ca binding at 37" was very rapid during the first 2-3 min. in both the 35% and the 3 5 4 5 % fractions, whereas during the following minutes there was only a minor increase (fig. 2). The Ca-binding capacity of the 3 5 4 5 % fraction after 10 min. incubation and was amounted to 4 . 7 k 1.5 nmol/mg_protein .
about half of that estimated in the 35% fraction, 8.5 0.9 nmol/mg protein. The Ca binding at different free CaZ+-concentrations.
It was of interest to investigate the C a binding of the microsomal fractions in relation to the free CaZ -concentration of the incubation solution. These experiments were performed at pH =6.8. As indicated in table 1 no Ca2+ was bound at 1 x lo-* M but there was a limited binding at 1.9~ M . The binding was increased with rising Ca2 concentrations up to a free Ca2 concentration of 5.8 x M. The microsomes were thus able to bind Ca at the Ca concentrations thought to be present in the myoplasm of smooth muscle. +
+
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2 1
8
6
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Fig. 2. The effects of ATP and temperature on the Ca binding of the 35% fraction (a) and of the 3 5 4 5 % fraction (b). The values represent the mean of 6 determinations S,E,M in the presence of o,35 mM ATP at 370;.--. in the presence of 0.35 m~ ATP at 4 ; .- x - x ~. in the absence of ATP at 37". ~
+
The ej'ect of oxalate on the Caz' -uptake. To skeletal muscle microsomes, added Caprecipitating anions, such as oxalate, increased the Ca uptake by precipitating Ca oxalate into the microsomal vesicles when the solubility product was reached (Hasselbach & Makinose 1963; Weber et al. 1966). The effect of oxalate on the rabbit colon microsomalCa uptake under standard conditions was studied in the concentration range of 0 to 5 mM. The Ca uptake of the 35% and 3 5 4 5 % fractions was stimulated at the highest concentration ( 5 mM) but not at the lower ones (fig. 3). The increase was evident after 5 min. At this point 5 m M oxalate increased the uptake of the 35% fraction by about 20% compared to the value obtained in the absence of oxalate (fig. 3a) and in the 3 5 4 5 % fraction the corresponding increase was about 90% (fig. 3b). In an incubation medium containing 5 m M MgATP and 5 m M oxalate the C a uptake of the 35% and 3 5 4 5 % fractions was initially rather slow, but after 3 min. rapidly increased. After this time it was almost linear with time up to a t least 20 min. (fig. 4), but still no equilibrium of the Ca uptake was reached. In the presence of 0.35 mM ATP and in the absence of oxalate the binding was maximal within 10 min. After 20 min. the Ca accumulation of the 35% and 3545% fractions in the presence of 5 mM MgATP
188
KARIN B. NILSSON ET AL. Table 1
The effect of the free Ca2+concentrations on the Ca binding of the microsomal subfractions. The microsomal fractions were incubated for 5 min. in solutions containing varying concentrations of ionized Caz+calculated according to Katz et al. (1970) by combining EGTA (ethyleneglycol-bis-(P-amhoethylether)-N,N-tetraacetic acid) with the total Ca at pH=6.8. The Ca added was measured with an atomic absorption spectrophotometer. Mean k S.E.M. n=6. Caz+ (MI
Total calcium (MI
EGTA (M)
1.ox 10-8
3.7x 1 0 - 5 3 . 6 1~0 - 5 4.0~ 9.0 x
0.57 x lo-, 0 . 5 9 ~10-3 0.79 x
1.2 x 1 0 - 7 1.9~ 5.8 x
~
and 5 mM oxalate was 5-7-fold that of the binding reached at 0.35 mM MgATP. Temperature.
The rate and the amount of Ca bound to the submicrosomal fractions was influenced by changes in the temperature. When the temperature of the standard incubation solution was reduced from 37" to 4" the amount of Ca bound by the 3 5 4 5 % fraction was markedly reduced (fig. 2b). There was no significant binding for the first 5 min. but after 10 min. some binding occurred. In the 35% fraction a binding of Ca occurred even during the first minute at 4" and this accumulation changed very little during the following 10 min. (fig. 2a).
Ca (nmol/mg protein) 35% 35-45% 0.0 0.013 k0.008 0.59 k0.29 3.90 k0.67
0.0 0.022 k 0.010 0.43 k0.18 3.98 k0.60
somewhat higher, 55.6f 3.9 nmol/mg protein. The hexosephosphate might thus be used for the regeneration of ATP. The time-response curve of the Ca binding of the 35% fraction indicated that the binding was a
b
The influence of ATP on the Ca binding.
The binding of Ca to the microsomal fractions was an energy dependent reaction. In the absence of exogenous ATP there was some binding of Ca to the 35% fraction after 1 min. but the binding did not increase with the time (fig. 2a). The 3545% fraction also bound some Ca in absence of exogenous ATP (fig. 2b). The question which arose was whether the binding was really independent of ATP or whether the microsomes eventually contained endogenous ATP and/or an ATP-generating system. Analysis of the microsoma1 fractions showed the presence of endogenous ATP in a concentration range of 4&70 nmol/mg protein. The glucose-6-phosphate (G6-P) content of the 35% fraction was 33.4& 1.7 nmol/mg protein. In the 3 5 4 5 % fraction it was
1 rnin
5 rnin
Fig. 3. The effect of different concentrations of Na,oxalate (CL5 mM) on the Ca uptake in the 35% fraction (a) and in the 3 5 4 5 % fraction (b) after 1 and 5 min. of incubation. Before the reaction was started by the addition of microsomal proteins (lo&] 50 pg) Na,oxalte (pH=7.2) was added to the test tube. The data represent the mean values of five to six different experiments S.E.M. The statistical significance of the differences between the paired samples in the presence and in the absence of Na,-oxalate is denoted by *= P~0.05.
*
189
CA-BINDING OF SMOOTH MUSCLE MICROSOMES
initially lower in the presence of 5 m M ATP than in the presence of 0.35 mM ATP (fig. 5a). After 10 min. no equilibrium was reached at the high ATP concentration. To determine the ATP optimum of the binding reaction in the absence of oxalate a short incubation time was chosen. The
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relationship between the Ca binding and the ATP concentrations (0-1.05 mM) was then investigated. A maximum of the Ca binding of the 35% fraction was observed at 0.05 m M ATP (fig. 5b). The 3545% fraction showed less variation in Ca-binding capacity a t different ATP concentra-
i4
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O
12 16 12 16
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I
20 min b
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Fig. 4. Ca binding and Ca uptake in the 35% fraction (a) and in the 3545% fraction (b). The values are the S.E.M. in average of six different experiments the presence of 0.35 mM MgATP and without Na,oxalate, in the presence of 5 mM-MgATP and 5 mM-Na,-oxalate.
+
0.2
aL
0.6
a8
1.0
mM ATP
Fig. 5. a) The Ca binding of the 35% fraction in the presence of 0.35 mM-ATP (.--.) and in the presence of 5 mM ATP (.--.). The data represent the mean of S.E.M. b) The effect of ATP 6 different experiments (0-1.5 mM) on the Ca binding in the 35% fraction (.--.) and in the 3545% fraction (.--.). Before the incubation of the samples was started by addition of microsomal proteins different concentrations of MgATP (adjusted to pH=7.2) were added to the incubation medium. The fractions were incubated for two min. and three min. respectively. The data represent the mean of five to six separate determinations S.E.M.
+
190
KARIN B. NILSSON ET AL. Table 2
Influence of salyrganand sodium azide o n the Ca binding of the microsomal subfractions. 1) = Salyrgan and sodium azide were added simultaneously with the incubation medium containing MgATP. 2) = The microsomal proteins were pretreated for 5 min. by salyrgan before addition of the incubation medium. Mean k S.E.M. n=2-6. Ca binding (nmol/mg protein/lO min.)
Microsomes
35%
3545%
Control 3 mM salyrgan” 3 mM salyrgan’’ 5 mM-NaN,”
6.0k0.8 2.8k0.6
4.9k0.7 1.8 k0.6
0 6.4+ 1.0
0
3.9k0.5
tions. A maximal response seemed to be obtained at about 0.35 m M ATP under our experimental conditions (fig. 5b). Since very low concentrations of ATP stimulated the C a binding, the possibility had to be considered that endogenous ATP present in the microsomes participitated in the binding in the absence of exogenous ATP. However, this question needs further investigation. Calculated from the intact rabbit colon preparation the concentration of ATP in the water phase of the muscle was estimated to be about 0.3-1.5 m M (Anderson & Mohme-Lundholm 1970), a value which decided us to choose 0.35 mM ATP in the standard incubation medium.
Effect of salyrgan and sodium azide on the Ca binding. Salyrgan (3 mM), an inhibitor of microsomal Ca accumulation, reduced the Ca binding of the 35% and 3 5 4 5 % fractions t o 52% and 60% of the control values respectively, when the drug was added simultaneously with ATP. When the microsomal proteins were pretreated for 5 min. by salyrgan before the addition of the incubation medium containing ATP, the Ca binding was completely inhibited (table 2). The addition of 5 mM sodium azide, a n inhibitor of mitochondrial Ca accumulation did not influence the C a binding of the 35% or 3545% fractions (table 2).
Ca binding of the mitochondria1 fraction. It has not been the aim of this study to investigate the Ca-binding properties of the mitochondrial fraction. It was found, however, that the 3 5 4 5 % subfraction contained some activity of cytochrome c oxidase and whole as well as damaged mitochondria were observed on the electron micrographs (Nilsson et al. 1977b). For purposes of comparison the Ca binding of the mitochondrial fraction was studied under the same experimental conditions, with the same incubation medium used in the investigations on the microsomal subfractions. The C a binding of the mitochondrial fraction was only observed in the fresh preparation and was lost rapidly during storage (fig. 6). After storage for 24 hrs at 4” no C a binding was detectable. Sodium azide (5 mM), an inhibitor of mitochondrial Ca accumulation, reduced the Ca binding of the fresh mitochondrial fraction by about 40%. Since the C a binding of the microsomal fractions was always investigated after cold storage for 20-24 hrs the contribution of mitochondria or mitochondrial fragments t o the C a binding of the 3 5 4 5 % fraction was probably insignificant under our experimental conditions.
Discussion Fig. 6 . The effect of storage (C24 hrs) on the Ca binding of the mitochondrial fraction. The Ca binding was studied in the same incubation medium as used for microsomal subfractions.
In this study it is shown that the crude microsomal fraction of the rabbit colon muscle can be resolved into three subfractions. Our results indicate
CA-BINDING OF SMOOTH MUSCLE MICROSOMES
that the microsomal subfractions isolated from rabbit colon smooth muscle differed somewhat regarding their Ca-binding capacity. The Cabinding per mg protein of the 35% fraction was usually greater than that of the 3 5 4 5 % fraction, when investigated under our standard conditions. There were also indications that the Ca binding of the 35% fractions was more influenced by the variation in the ATP concentration than that of the 3 5 4 5 % fraction (fig. 5). Beta-adrenergic agonists increased the Ca binding and cholinergic agonists released Ca from the 3 5 4 5 % fraction, whereas these drugs had no or very weak effects on the 35% fraction (Anderson et al. 1975; Nilsson & Anderson 1977; Nilsson et al. 1977). Differences were also observed in the activities of some enzymes as well as in the morphological pictures of the two fractions. These observations will be discussed in a subsequent paper (Nilsson et al. 1978). When combining these findings the 350/, fraction seemed to consist mainly of vesicles, which may have their origin from the plasma membrane as well as from the sarcoplasmic reticulum. The 3 5 4 5 % fraction probably consisted of fragments of the sarcolemma and some mitochondria1 fragments. The mitochondria were probably not involved in the Ca binding of the 3 5 4 5 % fraction under our experimental conditions, as the mitochondria completely lost their Ca-binding capacity after storage for 24 hrs at 4" (fig. 6). We also observed that sodium azide did not inhibit Ca binding of the 3 5 4 5 % fraction or in the 35% fraction (table 2). There are other observations which indicate the presence of microsomes with different Ca-binding properties in smooth muscle. Thus from vascular smooth muscle some investigators have isolated oxalate-sensitive microsomes (Fitzpatrick et al. 1972; Hess & Ford 1974; Shibata & Hollander 1974; Ford & Hess 1975; Webb & Bhalla 1976) while others found the microsomes to be oxalateinsensitive (Allen 1973; Baudouin-Legros & Meyer 1973; Zelck et al. 1975). Zelcket al. (1975), applying identical experimental techniques in studies on microsomes from pig coronary artery and from guinea pig ileum, found that the first mentioned microsomes were oxalate-sensitive whereas the others were not.
191
Our results show that the MgATP concentration is of importance for demonstrating convincing effects of oxalate. At high MgATP concentrations oxalate stimulated the Ca uptake about 3fold in the two microsomal subfractions (fig. 3,4), while only minor effects were observed at the lower MgATP concentrations (fig. 3). The Ca uptake in colon microsomes was stimulated by 5 m M oxalate; this concentration was also needed for stimulating the Ca uptake in vascular smooth muscle microsomes (Ford & Hess 1975). The relationship between Ca binding and ATPconcentration wascomplex. Most research workers (Batra & Daniel 1971a; Fitzpatrick et al. 1972; Baudouin-Legros & Meyer 1973; Hess & Ford 1974; Shibata & Hollander 1974; Zelck et al. 1974; Tomiyama et al. 1975; Wei et al. 1976) have investigated the Ca accumulation of smooth muscle microsomes at 3-5 m M ATP, which is about 8-fold the mean concentration of the intact intestinal and vascular smooth muscle (Anderson 1973 ; Anderson & Mohme-Lundholm 1970). The time course of the Ca accumulation of the 35% microsomes was not equal when studied at 0.35 and 5 mM ATP (fig. 5a). At the lower concentration most Ca binding took place within 2-3 min. whereas at the higher concentration the Ca uptake was initially rather slow but then increased gradually, but no equilibrium was reached within 10 min. The latter type of timecourse has been observed by those investigators who have used high ATP concentrations (Batra 1972; Fitzpatrick et al. 1972; Hurwitz et al. 1973; Hess & Ford 1974; Tomiyama er al. 1975). The time-course of the Ca accumulation in the presence of 0.35 m M ATP might better reflect the changes in cytoplasmic Ca concentration of colon muscle during relaxation than the more artificial medium containing 5 mM ATP and oxalate. Most of the binding of Ca occurred within the first minute, whereas in the intact colon muscle, for instance, P-adrenoceptor agonists require 30-60 sec. for complete relaxation (Anderson & MohmeLundholm 1970). The processes that lead to Ca binding in smooth muscles are still incompletely investigated. The cyclic AMP-protein kinase system has been suggested to be of importance for the P-adrenoceptor-
192
KARIN B. NILSSON ET AL.
stimulated Ca binding t o microsomal fractions (Andersson et al. 1976a). In other studies (Andersson el al. 1976b) the ATP-dependent Ca accumulation of colon microsomes have been studied by the so-called ESCA-technique (Electron Spectroscopy f o r chemical analysis). By this technique it was shown that phosphorous (probably in phosphate-form) was incorporated into the microsomal membranes along with calcium, when the microsomes were in a medium containing MgATP. Thus one possible mechanism for microsomal Ca binding might be phosphorylation of microsomal membranes. Another part of the ATP-dependent Ca accumulation in colon microsomes which was stimulated by oxalate may be similar to the “Ca pump” observed in sarcoplasmic reticulum isolated from skeletal muscle (Weber 1966). Acknowledgements Financial support was provided by the Swedish State Medical Research Council (B76- 14X-0208010A, B76-04X-04498-02, B76-04X-00101-12A), the Magnus Bergvall Foundation a n d the Swedish Society of Medical Research. We a r e indebted t o Mrs. L. Mackerlova and M r . S. Borjesson for valuable technical assistance.
Allen, J. C. : Characteristics of a vesicular Ca’ binding fraction from canine aorta: aortic relaxing factor (ARF). Biophys. J . 1973,13, 103a. Andersson, R.: Role of cyclic AMP and C a + + in mechanical and metabolic events in isometrically contracting vascular smooth muscle. Acta physiol. scand. 1973,87, 84-95. Andersson, R. G. G., L. Djarv, K. Nilsson & J. Wikberg: The role of calcium and cyclic nucleotides in the regulation of tension in smooth muscle of the gastrointestinal tract. In : Stimulus-secretion coupling in the gastrointestinal tract. Eds.: Case & Goebell, Medical Technical Publ. Co., Lancaster, 1976a, pp. 1-15. Anderson, R. & E. Mohme-Lundholm: Metabolic actions in intestinal smooth muscle associated with relaxation mediated by adrenergic a- and P-receptors. Actaphysiol. scand. 1970, 79, 244-261. Anderson, R. & K. Nilsson : Cyclic AMP and calcium in relaxation in intestinal smooth muscle. Nature New Bid. 1972,238, 119-120. Andersson, R. G. G., K. B. Nilsson, K.-E. Magnusson & L. Johansson : Relationship between calcium accumulating structures and the cyclic AMP system in +
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