Acta physiol. scand. 1978. 104. 68-73 From the Department of Obstetrics and Gynecology and the Department of Pharmacology, University of Lund, Sweden

Studies on calcium uptake by myornetrial microsomes with particular reference to the dependence on inorganic phosphate and oxalate BY

S. BATRA Received 13 February 1978

Abstract BATRA,S . Studies on calcium uptake by myornetrial microsomes with particulur reference :o the dependence on inorganic phosphate r i d oxalate. Acta physiol. scand. 1978.104.68-73. Ca uptake by microsomes isolated from non-pregnant rabbit myometrium was potentiated by both inorganic phosphate (Pi) and oxalate anions. Both Pi and oxalate bad little effect on the initial rate of uptake but a pronounced effect on the capacity of Ca uptake measured after 20 min which was greater in the presence of oxalate than that of Pi ( 5 mM each). The presence or absence of sucrose in the uptake medium had a significant effect on oxalate-induced potentiation of Ca uptake but not on that potentiated by Pi or that measured in the absence of either potentiating anion. A part of Ca accumulated additionally under the influence of sucrose could be removed by washing microsomes with KCI. Another significant difference between the pontentiating effect of oxalate and Pi was observed when the pH of the incubation medium was varied. In the presence of oxalate the pH optimum was between 6.46.8, whereas that in its absence or in the presence of Pi the optimal pH was around 7.2. Reduction in pH from 7.2 t o 6.8 along with the substitution of KCI by sucrose resulted in 3-fold increase in Ca uptake when oxalate was used as the potentiating anion. The results suggest that Ca is taken up by a different mechanism in the presence of oxalate than that i n its absence or when oxalate anion is substituted with inorganic phosphate.

Since the demonstration of an ATP-dependent Ca uptake in isolated microsomal and mitochondria1 fractions from the myometrium (Batra and Daniel 1969, Batra and Daniel 1971 a, Carsten 1969, Batra and Daniel 1971 b) Ca uptake by subcellular fractions isolated from several types of smooth muscles has been reported (see Janis and Daniel 1977 for references). Ca uptake by the microsomal fraction of smooth muscle differed not only quantitatively from that of skeletal muscle (Batra 1975) but certain qualitative differences, in particular the lack of stimulation by oxalate of Ca uptake by smooth muscle microsomes, were also noted (Batra and Daniel 1971 a, Baudovin-Legros and Meyer 1973, Zelck et al.

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1975, Worcel et al. 1976). In some other studies, however, oxalate stimulation in niicrosomes isolated from certain smooth muscles (Fitzpatrick eta[. 1977, Hurwitz et a/. 1973, Ford and Hess 1975, Godfraind et al. 1976, Raeymaekers et al. 1977) could be demonstrated. Although Ca uptake in some of these recent investigations (Ford and Hess 1975, Godfraind et al. 1976, Rayemaekers et al. 1977) was measured by Millipore-filtration technique (Martonosi and Feretos 1964), the retentate (material on the filter) rather than the filtrate was counted for Ca45-radioactivity determinations. This modification, which has also been recently made in the measurements of mitochondria1 Ca uptake (Jacobus et al. 1975) makes an important difference in that Ca uptake without unspecific (or loose) binding is measured. This is achieved by washing the material on the filters before the determination of the retained isotopic Ca. Furthermore, when low amounts of Ca are taken up, the reliability of the method measuring uptake directly in the retentate, rather than by the difference in the filtered and unfiltered samples, is better. Since inorganic phosphate (P,) is a physiological anion, occurring in relatively high concentrations in the myoplasm, we have, in the present study, examined its effects on Ca uptake kinetics in rabbit myometrial microsomes and compared them with those of oxalate. Not only we are able to clearly demonstrate the potentiation by P, as well as by oxalate but provide some evidence showing that the two anions increase Ca accumulation by different mechanisms.

Methods Preparations of microsomes

Nan-pregnant New Zealand white rabbits weighing between 2.7 to 3.2 kg were killed by cervical dislocation. The uterine horns were removed and placed i n Krebs-Ringer bicarbonate-solution bubbled continuously with 95s" 0, and 5:: CO,. The composition of the Krebs-Ringer medium was (mM), 115 NaCI, 4.63 KCI, 0.1 CaCI,, 1.16 MgSO,, 1.16 NaH,PO,, 21.9 NaHCO, and 49 m M glucose. Each horn was trimmed of excess fat and connective tissue, cut open lengthwise and endometrium removed by scraping while keeping the tissue in bubbling Krebs-Ringer solution throughout this period. The scraped myometrium was blotted dry and placed in ice-cold sucrose (0.25 M ) and Hepes (10 mM) solution, p H 7.2. The tissue, after weighing, was homogenized in about 10 volumes of the above sucroseHepes solution with a Polytron homogenizer for 10 s ' 3 periods (per g tissue) with intermittent pauses of 20 s. The homogenate was centrifuged at 1 000 v g for 10 min and the pellet discarded. The supernatant was filtered through 3 layers of gauze and centrifuged a t 15 000 x g for 15 min. The resulting pellet, which was enriched in mitochondria was occasionally saved for experiments dealing with Ca uptake measurements in mitochondria. The above supernatant was centrifuged at 4 0 0 0 0 x g for 1 h to obtain the microsoma1 pellet. The pellet was suspended in sucrose-Hepes solution to give a protein concentration of 1-2 mg/ml and used for Ca uptake experiments immediately. Protein concentration in the microsomal fractions was determined by the method of Lowry sr a/. (1951). Measurement of Ca uptake

Microsomes were incubated at 37°C in 1 ml of a solution containing (mM) 20 Hepes buffer (pH 7.251, 4 ATP, 5 MgCI,, 5 NaN, and 0.025 CaCI, including Ca45. The medium was supplemented with sucrose 0.2 M or KCI 0.1 M or NaCl 0.1 M. Microsomal protein concentration was 80-120 j'g per ml. After incubation, 0.2 ml of the reaction mixture was filtered at various times under suction through Millipore filters (HAWP 04500). The filters were subsequently washed with 2 ml of Hepes buffer containing sucrose o r KCI or NaCl depending on the composition of the reaction mixture. The radioactivity retained by the filters was counted by liquid scintillation spectrometry using Aquasol (New England Nuclear Corp.) as the scintillation fluid. Control tubes with the complete incubation mixture but without the microsomal protein were incubated in each assay. Control filters bound less than 0.05":, of the CaJ5 present in the reaction mixture irrespective of its composition.

70

S. BATRA

:.

p

30

j

w/

2

5

10

20

2

5

10

min

rnin

Fig. 1

Fig. 2

I'

20

Fig. 1 . Ca uptake by rabbit myometrial microsomes in the presence of 2 mM Pi (a), 5 mM Pi (A), 2 mM oxalate (n), 5 mM oxalate (A), and in the absence of potentiation anions ( 0 ) .Uptake medium was supplemented with KCI. Fig. 2. Ca uptake by rabbit myometrial microsomes from a medium supplemented with KCI ( 0 )or sucrose (0) of C a uptake from a medium supplemented with sucrose but microsomes after Millipore filtration were washed with KCI; see Methods.

(m). The middle curve represents data

Results The effect of P, on the time course of Ca uptake by rabbit myometrial microsomes is shown in Fig. 1. Whereas 2 mM oxalate had no significant effect on Ca uptake, the same concentration of PI increased Ca uptake slightly but significantly. Both oxalate and P, had little effect on the initial rate of uptake but a pronounced effect on the capacity of Ca uptake, measured after 20 rnin, and Ca uptake after this period was greater in the presence of 5 mM oxalate than that of 5 mM PI. When NaN, was omitted from the medium, Ca uptake increased by only about 20% (not shown). The presence of sucrose in the uptake medium had a significant effect on oxalate-induced potentiation of Ca uptake but not on that potentiated by P, or on that measured in the absence of either potentiating anion (Table I). There was no significant difference in Ca uptake when the medium was not supplemented or, was supplemented instead of sucrose with KCI or NaCl or choline chloride. This was true in both the absence and presence of potentiating anions indicating that sucrose increased potentiation by oxalate rather than the cations depressed it. TABLE I. The effect of sucrose or electrolytes added as supplements to the reaction mixture on Ca uptake by rabbit myometrial microsomes. Ca uptake was determined after 20 min of incubation. Values are means F S.E. of those obtained in 4 or 5 separate experiments.

Supplement

None Sucrose KCI NaCl Choline chloride

Ca uptake (,umol/g protein) without oxalate

with oxalate

with Pi

7.8853.0 10.66+ 1 . 3 8.36+ 1.2 9.57+ 0.9 12.88+ 1.2

23.15k2.1 58.46k4.8 20.39 & 2.4 19.96+ 2.1 19.51+1.7

21.501- 2.4 26.1612.3 21.42+ 2.0 22.38k2.3 26.54k2.4

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I

6.4

6.8

7.2

7.6

p H 7.2

pH6 8

PH

Fig. 3

Fig 4

Fig. 3. Dependence on p H of Ca uptake in the absence of potentiating anion ( 0 ) .or in the presence of Pi (a)or oxalate (W). Fig. 4. Effect of pH on oxalate-potentiated Ca uptake from a medium supplemented with KCI (open bars) or sucrose (closed bars).

Microsomes that had accumulated Ca from a sucrose containing medium in the presence of oxalate lost some of the bound Ca when washed with KCl instead of sucrose solution (Fig. Z), but the amount of Ca remaining in the microsomes was still considerably higher than that found after its accumulation from a KCl medium. Another significant difference between the potentiating effect of oxalate and PI was observed when the pH of the incubation mixture was varied (Fig. 3). In the absence of potentiating anions a pH change between 6.4 to 7.6 had little effect on Ca uptake. In the presence of P,, the maximum uptake was observed at pH 7.2 although the pH-optimum had a relatively broad peak. When oxalate was used as a potentiator of Ca uptake, the pHoptimum was 6.8 but even at 6.4 the uptake was near maximum. However, at pH 7.2 Ca uptake decreased by about 50% and was not significantly different than that observed with P, at this pH. Increasing pH to 7.6 further reduced oxalate-supported Ca uptake. Having observed that a reduction in pH or the inclusion of sucrose in the medium increased oxalate-supported, but not PI-wpported, Ca uptake considerably, the effect of sucrose at lower pH (6.8) was examined. As seen in Fig. 4, lowering the pH had an additive effect to that observed by inclusion of sucrose in the medium. Thus, a reduction in pH from 7.2 to 6.8 along with the substitution of KCI by sucrose almost trippled the amount of Ca taken up.

Discussion The present results clearly demonstrate a potentiation by both oxalate and Pi of Ca uptake by myometrial microsomes. Rabbit myornetrial microsomes prepared and assayed in the way as described here were also found to have much higher affinity for Ca uptake (Batra 1977) than that reported previously by us (Batra 1973, Batra 1975). Furthermore, a Castimulated ATPase activity, related stochionietrically to Ca uptake, could also be demonstrated (to be published).

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The potentiating effect of P, on Ca uptake by the microsomal fraction of smooth muscle was not unexpected but has not been demonstrated previously. The present data clearly show that potentiation by P, of Ca uptake differs not only quantitatively but also qualitatively from that observed with oxalate. Oxalate potentiation of Ca uptake in contrast to that by P, was sensitive to the composition of the medium as it was highly stimulated by the presence of sucrose in the medium as well as by lowering of pH below neutrality. The effect of increasing H+ ion concentration and the presence of sucrose were additive. Whether this stimulation is simply due to a greater permeability of oxalate to microsomal membranes under these conditions or whether it involves a more complicated mechanism cannot be known. In any event the present results showing a pronounced stimulation of Ca uptake by sucrose or by a reduction in pH specifically in the presence of oxalate might provide an explanation for some of the controversy on the oxalate potentiation of Ca uptake in smooth muscle microsomes. Our results on the pH influence on oxalate stimulated uptake are in agreement with those recently published by Codfraind et al. (1976) on intestinal smooth muscle microsomes. Krall et al. (1976) using a relatively alkaline (pH 8) reaction mixture recently reported oxalate potentiation of Ca uptake by rat myometrial microsomes. However, the rates of Ca uptake in their study were extremely low and uptake did not reach a plateau even after 30 min of reaction which is in contradiction with our previous data (Batra and Daniel 1971 a) as well as with those of Nishikori et al. (1977) reported recently. In fact, the data of Krall et al. showed practically no ATP-dependent uptake in the first 5 min whereas the data of other two studies mentioned above (Batra and Daniel 1971 a, Nishikori ei al. 1977) showed that Ca uptake reached a plateau after 5 min. In view of this it seems difficult to correlate the kinetics of Ca uptake presented by Krall et al. with the physiological function. No explanation was given by the authors for either these extremely slow rates of Ca uptake or the use of a reaction medium at pH 8. The present results also showed that a part of the Ca taken up under the influence of sucrose in the presence of oxalate could be released by KCI, but not by sucrose, washing. This would indicate some Ca binding at the surface of the vesicles for which K+ can compete with Ca++. However, there is a fraction of Ca which is probably transported inside the vesicle under the influence of sucrose as KCI washing removed only 39% of additionally accumulated Ca. The study of the mechanism of this action of sucrose deserves further attention. Interestingly, sucrose has been shown to antagonize the inhibition by certain drugs and elevated temperature of Ca uptake by cardiac sarcoplasmic reticulum (Solaro et al. 1972). Since not only the effect of pH but also of sucrose was specific for oxalate, it is suggested that Ca is taken up by a different mechanism in the presence of oxalate than in its absence or when oxalate anion is substituted with inorganic phosphate. I am grateful to Miss Lena Timby for her excellent technical assistance. This work was supported by the Ford Foundation and the Swedish Medical Research Council (project No. 4504).

References BATRA, S., The role of mitochondria1 calcium uptake in contraction and relaxation of the human myometrium. Biochim. biophys. Acra (Amst.) 1973. 305. 4 2 8 4 3 2 .

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BATRA,S., The role of mitochondria in the regulation of myoplasmic calcium concentration in smooth muscle. In: Calciunt Transport in Contraction and Secretion. Eds. E. Carafoli, F. Clenienti, H. Drabikowski and A. Margreth, North-Holland, Amsterdam. 1975. 87-94. BATRA,S., T h e importance of calcium binding by subcellular components of smooth muscle in excitation contraction coupling. In: E.wcitation-corrtrn~/ionCoupling in Smooth Muscle. Eds. R. Casteels, T. Godfraind and J . C. Ruegg, 1977. 225-237. North-Holland, Amsterdam. BATRA,S. C. and E. E. DANIEL,Ca uptake by mitochondria1 and microsomal fractions of myometrium from estrogen-dominated rats. Proc. I V int. Congr. Pharmacol. 1969. p. 18 I . BATRA,S. C. and E. E. DANIEL,Effect of multivalenl cations and drugs on Ca uptake hy the rat myometrial microsomes. Comp. Biochem. Plij~siol.197 1 a. 38. 285-300. BATRA,S. C. and E. E. DANIEL,ATP-dependent Ca uptake in subcellular fractions of uterine smooth muscle. Conip. Biocheni. Physiol. 1971 b. 38. 369-385. M. and P. MEYER,Effects of angiotensin, catecholamines and cyclic A M P on calcium BAUDOLJIN-LEGROS, storage in aortic microsomes. Brit. J. Plinrniticol. 1973. 47. 377-385. 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. and L. HURWITZ,A calcium pump in vascular smooth FITZPATRICK, D. F., E. J. LANDON,G. DEBRAS muscle. Science 1972. 176. 305-306. FORD,G. D. and M. L. HESS,Calcium accumulation properties of subcellular fractions of bovinevascular smooth muscle. Circular. Res. 1975. 37. 580-587. and N. VERHEKF, Calcium incorporation by smooth muscle microsomes. GODFRAIND, T., X. STURBOIS Biochim. biopliys. Acta (Amst.) 1976. 455. 254-268. G. DERBAS and E. J. LANDON,Localization of calcium pump activity HURWITZ,L., D. F. FITZPATRICK, in smooth muscle. Science 1973. 179. 384-386. and E. CARAFOLI, Aspects of energy-linked calcium JACOBUS, W. E., R. TIOZZO,G. LLIGLI,A. L. LEHNINGER accumulation by rat heart mitochondria. J. h i d . Chem. 1975. 250. 7863-7870. JANIS,R. A. and E. E. DANIEL,Ca2+ transport by subcellular fractions from smooth muscle. In: The Biochemistry of Smooth Muscle. Ed. N. L. Stephens, 1977. 653-671. University Park Press, Baltimore. and S. G. KORENMAN, Hormonal control of uterine contraction: CharacterizaKRALL,J. F., J. L. SWENSEN tion of cyclic AMP-dependent membrane properties in the myometrium. Biochirn. bioplivs. Acta (Amst.) 1976. 448. 578-588. A. L. FARRand R. J. RANDALL,Protein measurement with the folin LOWRY,0. H., N. J. ROSEBROUGH, phenol reagent. J . biol. Chem. 1951. 193. 265-275. Sarcoplasmic reticulum. I. The uptake of Cat+ by sarcoplasmic reticulum MARTONOSI, A. and R. FERETOS, fragment. J. biol. Chem. 1964. 239. 648-658. K., T. TAKENAKA and H. MAENO,Stimulation of microsomal calcium uptake and protein NISHIKORI, phosphorylation by adenosine cyclic 3',5'-monophosphate in rat uterus. Molec. Pharrnacol. 1977. 13. 671-678. S. BATRAand R . CASTEELS, A comparative study of the calcium accuniulaRAEYMAEKERS, L.. F. WUYTACK, tion by mitochondria and microsomes isolated from the smooth muscle of the guinea-pig taenia coli. Pfliigers Arch. ges. Physiol. 1977. 368. 217-223. SOLARO,R. J., E. W. GERTZand F. N. BRIGGS.Sucrose antagonism of drug and temperature effects o n . (Amst.) 1972. 255. 751-761. cardiac sarcoplasmic reticulum. Biochinr. b i o p h ~ ~ sActa WORCEL,M., A. PAPADIMITRIOU, G. HAMONand P. K. RANGACHARI, Role of transmembrane Ca2+movements and Ca2+binding in the activation of rat uterus smooth muscle contraction. In: Smooth Muscle Pharmacolog)i and Pliysiulogy. Eds. M. Worcel and G. Vassort, INSERM Symp., Paris. 1975. 353-362. ZELCK,U., U. KARNSTEDT and E. ALEIRECHT, Calcium uptake and Ca release by subcellular fractions of smooth muscle. Acta b i d . med. germ. 1975. 34. 981-986.