The calcium uptake in smooth muscle microsomal vesicles is reduced by centrifugation

The calcium uptake in smooth muscle microsomal vesicles is reduced by centrifugation

Cell Calcium 5: 205-210, 1984 THE CALCIDMLIPTAKEIN SMXYI’HMUSCLEMICROSWALVESICLES IS REDUCED BY CENTRIFUGATION L. Raeymaekers and R. Casteels Lab...

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Cell

Calcium

5:

205-210,

1984

THE CALCIDMLIPTAKEIN SMXYI’HMUSCLEMICROSWALVESICLES IS REDUCED BY CENTRIFUGATION L. Raeymaekers and R. Casteels Laboratorium voor Fysiologie, University B-3000 Leuven, Belgium (reprint requests

of Leuven, to LR).

Campus Gasthuisberg

ABSTRACI A membrane fraction was isolated from the smooth muscle of the pig stomach by density gradient centrifugation. It was observed that the ATP-dependent Ca uptake in this fraction was diminished if the microsomes were pelleted by differential centrifugation. The decrease of the oxalate-independent Ca uptake was relatively small, but the oxalatestimulated Ca uptake was reduced dramatically. Evidence is presented which indicates that the selective decrease of the oxalate-stimulated Ca uptake is mainly caused by mechanical damage of the vesicles. Since the oxalate-stimulated Ca uptake can be largely preserved by avoiding pelleting during the membrane fractionation, this observation may be very useful for the further study of Ca transport in subcellular fractions of smooth muscle. IINTRODUCTION Ca transport in vesicular membranes isolated from smooth muscle is studied to elucidate the function and the characteristics of the different Ca transport systems in the living muscle cell (e.g. l-10). It is obvious that extrapolations from Ca uptake studies on isolated membrane fractions to Ca transport in the intact cell may be valid onIy if the functional properties of the membranes are not grossly altered during isolation. Especially ion uptake may be very sensitive to alterations of the membranes since it not only depends on the functioning of the ion transport carrier but also on the integrity of the vesicular membrane, which should be sufficiently impermeable to retain the transported molecules in the lumen of the vesicle. The Ca uptake by membrane vesicles isolated from smooth muscle is stimulated by oxalate. Evidence has been presented which indicates that the oxalate-stimulated Ca uptake occurs in vesicles derived from the endoplasmic reticulum, whereas the oxalate-independent Ca uptake occurs as well in endoplasmic reticulum as in plasma membrane vesicles (6,8,10).

205

It was recently observed that the oxalate-dependent Ca uptake was diminished during smooth muscle subcellular fractionation, but the mechanism responsible for this phenomenonwas not studied (11 ,I 2). We now report that the oxalate-stimulated Ca uptake is very sensitive to pelleting by centrifugation. METHODS The smooth muscle of the antral part of the pig stomach was finely minced by passing through a press as previously described (13) . The mince was dispersed in 7 volumes of 0.25 M sucrose containing 0.5 mM phenylmethylsulfonylfluorid and 1 till dithiothreitol and further homogenized with a Polytron homogenizer at maximumspeed for 30 set, separated in three bursts of 10 sec. The homogenate was centrifuged in a Sorvall GS3 rotor at 9000 rev./min for 20 min. The pellet and the floating fluffy layer were discarded. Membranefractions were isolated from the supernatant by floating them into a sucrose gradient, extending from 45 % to 15 % layered on top of the supernatant. Solid sucrose and KC1were added to the supernatant to obtain final concentrations of 50 % (w/w) and 0.6 M respectively. Centrifugation was carried out in a BeclonanTi 15 zonal rotor at 32.000 rev./min for 20 hours. The rotor was filled with 260 ml overlay of 8 % sucrose, 400 ml sucrose gradient and 1000 ml supernatant. Tine sucrose gradient solution contained 0.6 M KC1 and 1 &I dithiothreitol. Fractions were collected in 20 ml portions. For the present experiments, the membranesequilibrating at 20-24 % sucrose were selected because this material was most enriched in Ca uptake activity (an extensive analysis of all fractions will be published later). Probably, this is not a pure fraction but a mixture of membranesderived from intracellular membranesand plasma membranes. The membranefraction was diluted slowly with 4 volumes 0.6 M KC1 containing 1 mMdithiothreitol. This diluted fraction will be designated as “control fraction” . In order to study the effect of centrifugation on the Ca uptake, the membraneswere either pelleted or sedimented on a sucrose cushion in a BeckmanTi60 rotor. The sucrose cushion was 5 ml of a solution containing 30 % sucrose (w/w) , 0.6 M KC1 and 1 II&Idithiothreitol. In some experiments an intermediate layer of 15% sucrose was applied between the 30% sucrose cushion and the vesicular suspension in order to obtain a more complete separation between the banded vesicles and the supernatant. The pellets were resuspended in 4% sucrose, 0.6 M KCl, 1 mMdithiothreitol by using a Potter-Elvehjem homogenizer (always the same one) at 200 rev./min, 10 strokes up and down. In most experiments the control fraction and the fraction recovered on the sucrose cushion were subjected to the same resuspension procedure using the Potter-Elvehjem homogenizer. The 45Ca uptake was measured by Millipore filtration on the same day the fractions were obtained from the gradient. The 45Ca uptake was started by adding 1 volume vesicle suspension to 3 volumes Ca uptake solution at 37’C. The composition of this solution was such that the following final concentrations were obtained (mM): KCL 150, Na-azide 5, Imidazole HCl (pH 6.9 at 37’C) 25, Tris-ATP 5, MgC125, Ca-EGTA1. It also contained some residual sucrose from the membranesuspension. When indicated, the mediumwas supplemented with 5 &I K-oxalate. Ca bound

in the absence of ATP was subtracted to correct for ATP-independent Ca binding. The oxalate-stimulated Ca uptake is defined as the amount of Ca retained on the filters after 20 min incubation in the presence of 5 til oxalate minus the amount of Ca retained in the absence of oxalate. Protein was measured by the method of Lowry et al. (14) following precipitation with trichloroacetic acid to remove substances which coulJ interfere with the color development. RESULTS The time course of the Ca uptake in the membrane fractions used in this study follows the typical pattern observed on smooth muscle subcellular membranes. In the absence of oxalate, the Ca uptake reaches a steady state value after 2 to 5 minutes and the plateau value of this Ca uptake changes very little with prolonged incubation. In the presence of oxalate the Ca uptake increases with time. In our present conditions the Ca uptake in the presence ofoxalateproceeded linearly with time for at least 30 min. In the course of an investigation of the distribution of the Ca uptake in membrane fractions separated by sucrose density centrifugation, it was observed that there was a large difference in the rate of Ca uptake in the presence of oxalate between material obtained directly from the gradient and the same material which had been concentrated by pelleting. In order to find out whether this reduction was caused by centrifugation or by some other factor, care was taken to treat all the samples in exactly the same way, except for different centrifugation conditions. In the membrane vesicles were also sedimented by addition to pelleting, centrifugation on a cushion of a denser sucrose solution. For each condition, three different centrifugal forces were selected. The values of the Ca uptake in the fractions obtained by these different procedures are shown in Table 1. The figures clearly show that pelleting causes a dramatic reduction of the oxalate-stimulated Ca uptake. Pelleting at 64,000 or 180,000 xg for 30 min reduced the specific oxalate-stimulated Ca uptake to 16 % of the control. This effect was much smaller if the membranes were recovered on a cushion of a denser sucrose solution. Under this condition it was reduced to only 60 “, of the control. In contrast, pelleting reduced the oxtilate-independent Ca uptake to only 68 90 of the control, and concentrating on a cushion of sucrose to only about 75 9,. The Ca uptake was further reduced by both centrifugation procedures if the centrifugation was carried out at high g force (250,000 x g for 60 min). Again the effect was more pronounced on the oxalate-stimulated Ca uptake than on the oxalate-independent uptake. For the oxalate-independent Ca uptake there was no significant difference between pelleting and concentrating on a cushion of sucrose, except for high g forces and prolonged centrifugation.

207

TABLE 1 The effect of centrifugation (pelleting or sedimenting on a cushion of a dense sucrose solution) on the oxalate-stimulated and oxalate-independent Ca uptake in a smooth muscle membrane fraction. The Ca uptake was determined after incubation for 20 min. The data are the percentage of’the Ca uptake observed in the control fraction. They are given as the mean ? S.E.M. of duplicate measurements on n preparations. The number of preparations is given in parenthesis. The values of 100 % in the control fraction correspond to 103 nmol Ca . mg-1 for the oxalateindependent Ca uptake and to 471 nmol . mg-’ for the oxalate-stimulated Ca uptake. The last column shows the ratio of the amount of Ca retained in the presence of oxalate to that taken up in the absence of oxalate . oxalate-stimulated Ca uptake (90)

control pelleted

fraction

100

oxalateindependent Ca uptake (90)

100

Fold of stimulation by oxalate

5.6

fractions

64,000 x g,, for 30 min

16.Okl.7

(4)

68

180,000 x g,, for 30 min

16.125.3

(5)

63.12

250,000 x g, for 60 min

4.lkO.5

(4)

60.327.4

180,000 x g,, for 30 min

57

250,000 X gav for 60 min

43.5

? 7.9

(4)

1.9

(4)

1.8

45.9kl2.6

(3)

1.3

(3)

73.7+15

(3)

5.7

(5)

76.4k11.5

(5)

4.0

(2)

67

(2)

3.0

9.8

fractions concentrated on a sucrose cushion 64,000 x g, for 30 min

28.2

208

DISCUSS ION The following mechanisms were considered to explain the loss of Ca uptake activity after centrifugation of the vesicles: 1) the loss of some soluble factors 2) mechanical damage caused by resuspending the pellet 3) osmotic chock caused by dilution of the density gradient fraction 4) the high hydrostatic pressure which develops during centrifugation 5) mechanical stress caused by compression of the vesicles against the wall of the tube and against each other during pelleting. A contribution of a loss of soluble factors to the reduction of the oxalate-dependent and -independent Ca uptake is very unlikely since the control material was prepared by centrifugation (floating) in a density gradient, a procedure which should remove soluble material from the membranes. However, if some soluble material would remain with the membranes, some of it would still be mixed with the vesicles at the interface of the sucrose cushion, whereas with the pelleting technique it would be decanted. In order to eliminate this possibility, an additional layer of sucrose was applied between the sucrose cushion and the suspension of vesicles to obtain a complete separation between the vesicles and the supernatant. This procedure did not affect the results. Possibility 2 can be excluded because the control fraction was subjected to the same procedure for resuspending as the pellets. Moreover? if it was not subjected to this treatment, it did not present a significantly higher Ca uptake (data not shown). Also explanation 3 does not apply since the control fraction was diluted in the same way as the centrifuged fractions. The high hydrostatic pressure during centrifugation (explanation 4) could contribute to the non-specific decrease of the Ca uptake, as has previously been observed on sarcoplasmic reticulum of skeletal muscle (15). However, it cannot explain the decrease of the oxalate-dependent Ca uptake in the pelleted fractions since the pellets and the fractions sedimented on a sucrose cushion are subjected to similar hydrostatic pressures. Therefore, mechanical stress remains as the most plausible factor to explain the selective decrease of the oxalate-stimulated Ca uptake during pelleting. These results show that the oxalate-stimulated Ca uptake in smooth muscle membranevesicles is very sensitive to damagewhen subjected to pelleting by centrifugation. Since pelleting was probably involved in all previous subcellular fractionations, the quantitative data on the capacity to take up Ca in the presence of oxalate by smooth muscle microsomes should be interpreted with caution. By modifying the centrifugation procedure, it is possible to reduce to a great extent the selective loss of the oxalate-stimulated Ca uptake during subcellular fractionation. This observation may be very useful for the further study of Ca transport in smooth muscle subcellular fractions and for improvement of the purification of vesicles by calcium oxalate loading.

We thank Dr. Heirwegh of the Laboratoy of Hepatology for the use of the Becknan Ti 15 rotor.

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Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry 193,265-275. S. and Guillain, F. (1981) Pressure-induced 15. Champeil, P. , Btichlen, inactivation of sarcoplasmic reticulum adenosine triphosphatase during high-speed centrifugation. Biochemistry 20, 1520-1524. Received 11/l/84 Revised version received and accepted 210

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