Calcium uptake into rat small intestinal brush border membrane vesicles: Characterization of transmembrane calcium transport at short initial incubation times

Cell Cahum (1987) 8, 157-169 8~8Longman Group UK Ltd 1987

CALCIUM UPTAKE INTO RAT SMALL INTESTINAL BRUSH BORDER MEMBRANE VESICLES: CHARACTERIZATION OF TRANSMEMBRANE CALCIUM TRANSPORT AT SHORT INITIAL INCUBATION TIMES W.E.J.M.

Ghijsen,

U. Ganguli,

Institute of Physiology, Winterthurerstrasse 190, to HM).

G. Stange,

University CH-8057

of Ziirich,

P. Gmaj Ziirich-Irchel, Switzerland

and H. Murer

(reprint

requests

ABSTRACT Calcium transport into brush border vesicles from rat small intestine was investigated by determining uptake rates at very short incubation periods. At incubation times up to 1 second a linear relationship between calcium uptake and time was observed at free calcium concentrations ranging from 1 UM to 5 mM. At time points above 1 second calcium uptake deviates progressively from linearity. Several lines of evidences (EGTA-wash, dependency on membrane potential, temperature sensitivity and effect of the calcium ionophore A23187) suggest transmembrane transport rather than extravesicular binding of calcium as being responsible for calcium uptake. Saturation experiments performed under initial linear and curvilinear uptake conditions show a saturable transport component in the umolar and only a tendency to saturate in It is concluded that uptake values far the mmolar concentration range. from equilibrium are characteristic for transmembrane flux of calcium. Transmembrane flux of calcium is mediated by multiple and potentialsensitive mechanisms.

INTRODUCTION Transcellular transport of calcium across the small intestinal enterocyte involves two different transmembrane transport steps (l-4,30) a) influx into the cell across the brush border membrane along a steep electrochemical gradient and b) efflux across the basal-lateral membranes against a steep electrochemical gradient. Studies lateral in the

with isolated brush border membrane vesicles and isolated basalmembrane vesicles have been used to characterize the mechanisms transmembrane movements of calcium. Studies with basolateral

157

membranes documented the existence of an ATP-dependent calcium transport system and provided predominantly .indirect evidence for a sodium/ calcium exchange mechanism (5-9). The studies with brush border membrane vesicles were difficult because only equilibrating calcium uptake occurred and because a major part of calcium represented binding of calcium - mainly to intravesicular sites (10,ll). Nevertheless, significant information on the brush border transport system for calcium was derived from studies with vesicles: it was reported that the transport system is saturable and under control of 1,25(OH)z Vit. Dg and corticosterone (lo-18,30). The kinetic parameters for calcium uptake by isolated brush border membrane vesicles were obtained by determinations approaching (e.g. 4, 29) or deviating considerably from initial linear uptake rates (e.g. 10, 12,16). Thus, it is difficult to decide by what extent the observed parameters are related to a transmembrane transport step or to intravesicular binding. This problem was the basis of the study described The time-dependency of brush border calcium uptake under condihere. tions far from equilibrium was analyzed to estimate real initial rates. A very rapid initial linear phase of calcium uptake could be dissociand transport and kinetic ated from a more prolongued curvilinear phase, parameters under both conditions were compared. MATERIALS

AND METHODS

Materials 45CaC1, wasobtained mycin was purchased at least analytical

from New England Nuclear (Dreieich, from Sigma (Miinchen, FRG). All other grade.

FRG). Valinochemicals were

Preparation of brush border membrane vesicles: Brush border membrane vesicles were prepared by a divalent cation precipitation method similar to that described earlier (19,201. Mucosal scrapings from mid jejunum of 2 male Wistar rats (180 - 200 gr.) were homogenized in 30 ml of a buffer containing 300 mM mannitol, 5 mM EGTA, 12 mM Tris adjusted with HCl to a pH of 7.1. After dilution with 120 ml of ice-cold distilled water, MgC12 was added to a final concentration of 10 mM and the The supernatant resulting homogenate was kept on ice for 15 minutes. from a 3000 g x 15 min. centrifugation was centrifuged at 27000 g for The pellet was resuspended in 30 minutes (Sorvall RC 5B, SS 34 rotor). 5 mM EGTA adjusted with 30 ml of a buffer containing 60 mM mannitol, a second addition of MgC12 (10 mM) the 2 Tris to a pH of 7.1. After were centrifugation steps (3000 x 15 min. and 27000 g x 30 min.) repeated. The pellet was resuspended in 30 ml of a buffer containing with Tris to a pH of 150 mM KCl, 1 m&l MgC12 and 20 mM HEPES adjusted 7.4. The final pellet obtained by a centrifugation at 27000 g for 40 minutes was resuspended in the KC1 buffer (see above) in a volume required for the further experiments (200 - 250 ~1). The final membrane in aminopeptidase M pellet was enriched approximatively 13- to 16-fold (data not shown, 20). Occasionally we have also isolated membranes from the duodenum. 158

Protein was measured by the Enzyme assays and protein determinations: Bio-Rad protein assay according to Bradford (21) using bovine plasma Aminopeptidase M activity which was used gamma globulin as a standard. as a marker for the brush border membrane was measured according to (22) using a LKB 8600 reaction rate analyzer equipped Haase et. al. with a kinetic data processor. The uptake of calcium was started by the addition Calcium uptake assay: of 10 - 20 1~1 of the membrane vesicle suspension to 100 - 200 ii1 of the incubation medium with a solute composition similar to the KC1 buffer used for vesicle suspension (150 mM KCl, 1 mM MgClz, 20 mM HEPES adjusted with Tris to a pH of 7.4) containing in addition 0,s mM EGTA, 0,s mM HEDTA and sufficient 45CaC12 (1 pCi/ml) to yield the desired concentration of free calcium (final concentrations after mixing of vesicle .suspen;Son with incubation medium). Although, EGTA and HEDTA buffer free Ca concentrations effectively in the micromolar range, these ligands were used in the entire calcium concentration range studied (up to 5 mM) to rule out possible differences in calcium uptake dye to these chelators. In some experiments Ca-ionophore A23187 or the K -ionophore valinomycin was added to the incubation medium at the concentrations mentioned in the legend of Table I. At different time intervals, a 20 1~1 sample was removed and diluted into 1 ml of an icecold stop solution containing the same solute composition as used for vesicle suspension and in addition 1 mM EGTA. The diluted uptake medium was immediately filtered through Sartorius filters (cellulose nitrate, 0.6 urn) kept under suction. All incubations (except 1 condition in table I: 37'C) were carried out at room temperature 20' - 22'C. For the rapid uptake measurements (below 15 seconds) a semiautomatic set up was used as described by Kessler et. al. (23) and now constructed by Innovativ AG (Adliswil, Switzerland): 20 Ul aliquots of vesicle suspension and 20 ~1 of incubation medium were mixed. The composition of the incubation medium was the same as described above for the longer incubations, but with 10 i.lCi 45CaC12 for the 0.5 and 1 set time points and 1.5 lXi for the longer time points up to 10 sec. After filtration the filters were washed with 3 ml of the ice-cold stop solution. The radioactivity retained on the filters was counted by liquid scintillation techniques. Unspecific binding of the isotopes was determined the corresponding amount of labelled incubation solution containing the vesicles at O°C and by The blank values were always less than 20 % of the lowest activity and were substracted. Single presented throughout the paper. All experiments three times with qualitatively identical results. experiment all determinations were performed at

by the addition of medium to the stop subsequent filtration. the uptake values with experiments will be were repeated at least Within the same least in triplicates.

In preliminary experiments, we have used 2 different stop solutions: stop solution containing EGTA (1 mM) and a stop solution containing 50 PM LaC13. In agreement with data published by Bikle et. al. (16) the uptake values were slightly lower by using the EGTA stop solution,

1.59

A

in addition a smaller experimental scatter was obtained by using EGTA stop solution. Therefore, we have used the EGTA-containing tion throughout this study.

the solu-

2+ Calculations of free Ca : The total CaC12 c;Tcentrations needed to obtain the desired concentrations of free Ca were calculated according to van Heeswijk et. al. (24) an by using published association constants for the ligands (25,26). Abbreviations: HEPES, 4-(2-hydroxyethyl)l-piperazineethane sulfonic acid; EGTA, ethylene glycol bis (P-aminoethyl ether)-N,N'-tetraacetic acid; HEDTA, N-hydroxyethyl ethylene diamine triacetic acid; TMA, tetramethylammonium

RESULTS Calcium uptake by brush border membrane vesicles was analyzed at different concentrations and for different incubation periods. In agreement with earlier publications on this subject (e.g. 10,16), calcium uptake equilibrated within 60 to 120 minutes in a curvilinear manner; the equilibrium uptake exceeded several fold the equilibrium space estimated by the equilibrium space for D glucose (data not shown). Calcium uptake was stopped with an EGTA-containing solution to complex extravesicular free and bound calcium. Moreover, replacement of EGTA by LaC13, which competes with calcium for extravesicular calciumbinding sides did not drastically change the calcium uptake values (see Methods). These results suggest that under the experimental conditions used the measured calcium uptake values reflect mainly calcium present in the vesicle interior. In an attempt to determine calcium uptake rates we analyzed the timedependency of calcium uptake in the vesicles at short incubation times 1 shows calcium uptake values measured at three up to 60 sec. Figure different Ca 2+ concentrations varying from 10e6 to 5.10d3 M. At all calcium uptake deviates fast from three calcium concentrations, linearity and is in fact only linear during the first second of one could discriminate between incubation (Figure 1B). Consequently, a linear initial uptake phase (up to 1 set) and a so-called curvilinear uptake phase between 1 set and 60 sec.

160

B)

2.0 ;;ii

/

1.5

F ’ 1.0 A 0”

5 1015

60

30

incubation

Figure 1. Initial concentrations:

time

course

cl 0.5 E c l&LLiiL

of

0 time

calcium

1

2

3

45

(seconds)

uptake

at

various

calcium

Membrane vesicles were suspended in the KC1 buffer (see Materials and Methods) and uptake of calcium was studied after addition of the vesicle suspension to an incubation medium consisting of the KC1 buffer containing 0.5 mM EGTA, 0.5 mM HEDTA and sufficient CaC12 to obtain the free concentrations as indicated in the figure. The uptake of calcium was measured with a semiautomatic set up as indicated in Materials and Methods. The values represent means of a typical experiment performed or in triplicates (above 1 set). The in quadruplicates (below 1 set) individual values were within a 10 % scatter. A) Uptake values from 0 to 60 set of incubation time; B) Uptake values from 0 to 5 set of incubation time.

The experiments summarized in Table I show that calcium uptake measured at a high substrate concentration (0.5 mM) and a low substrate concentration (1 PM) fulfills several criteria of transmembrane transport (see Discussion) both at a time point where linear uptake rates are present (1 set) and in the curvilinear phase of calcium uptake. Uptake of calcium is stimulated by increasing the incubation temperature from 20°C to 37'C. In addition, an inside negative membrane potential stimulates calcium uptake. The addition of the ionophore A23187 stimulates the initial calcium uptake rate 7- to 11-fold; this stimulation decreases at longer incubation times and is disappeared under equilibria1 uptake conditions, i.e. after 120 min incubation (results not shown). Furthermore, the addition of EGTA (0.5 mM) and of the ionophore A23187 leads to a time-dependent loss of calcium from vesicles loaded with calcium in the absence of the ionophore (data not shown; 10,27). 161

TABLE

I.

EFFECTS OF TEMPERATURE, MEMBRANE POTENTIAL AND CA-IONOPHORE A23187 ON CALCIUM UPTAKE AFTER DIFFERENT INCUBATION PERIODS

Membranes (5-10 mg/ml) were loaded with 150 mM KC1,20 mM HEPES adjusted with Tris to a pH of 7.4, 1 mM MgC12 and 10 ug valinomycin/ml. A23187 was added at a concentration of 10 ug/ 1 ml vesicle suspension. Uptake was initiated by mixing 1 volume (20 ~1) of membraneswithl volume (20 ~1) of incubation medium containing the same substances at identical concentrations and in a$l.ition 0.5 mM EGTA, 0.5 mM HEDTA and 45CaC12 to obtain a free Ca concentration of 1 I.rM or 0.5 m.M. For the Ki > Kg condition, we diluted 5 1~1 of membranes into 45 1.11 of incubation medium containing TMA-Cl instead of KCl. The 1 set uptake rate was obtained by using the rapid uptake apparatus, the 1 min uptake rates are obtained by taking the slopes of the 15 set, 30 set, 45 set and 60 set uptake values (linear regression). Except for the 37'C condition incubation temperature was 20°C. The values represent the mean values of a quadruplicate determination + standard errors obtained in a typical experiment. * Note the different-time units used for the initial and curvilinear calcium uptake rates.

A)

low

calcium

concentration:

1 W

initial linear *nmol/mg prot./sec control 2o" Ki

(2OOC)

+ 37Oc > Ko + Val

A23187

B) high

0.07 0.14 0.14 0.52

calcium

control

(2OOC)

0.26

2o" + 37Oc

0.56

Ki

0.46

> Ko + Val

A23187

3.26

rate % change

-+ 0.01 -+ 0.02

prot./sec

0.5

116

2.14

109

2.18

701

3.62

-+ 0.14 -+ 0.16 -+ 0.21 -+ 0.31

rate % change

112 116 258

mM free

schange

-+ 0.05 -+ 0.03 -+ 0.02 -+ 0.26

curvilinear *nmol/mg prot./min 1.01

-+ 0.p1 -+ 0.04

concentration:

*nmol/mg

free

*nmol/mg 4.42

prot./min

% change

115

15.16

-+ 0.3 -+ 0.6

76

8.16

-+ 0.3

85

1153

16.68

-+ 2.2

277

162

243

Measured as initial linear uptake rate at 1 second or measured as curvilinear rate between 15 seconds and 45 seconds of incubation, calcium uptake was non-linearly related to the extravesicular calcium concentration indicating saturability. When the data for the low concentration range are plotted separately (up to 100 PM, insets of Figure 2), a saturable component becomes apparent which would not be

B)

1

2

3

4

5

mM

mM calcium

Figure 2. curvilinear

Calcium brush

concentration

concentration-dependence border calcium uptake:

of

initial

linear

and

Uptake of calcium was determined under conditions identical to that given in the legend to Figure 1. Figure 2A shows the uptake values after 1 set of incubation; Figure 2B shows the uptake values extrapolated by linear regression to a 1 minute incubation period on the basis of the 15 set, 30 set and 45 set incubation time points. The values represent mean values of a typical experiment performed in quadruplicates for the 1 set time points or in duplicates for the longer incubation periods.

observed in a more extended concentration range (Figure 2). Based on this observation we have estimated the kinetic parameters for the calcium uptake for short incubation periods (1 set) and prolongued incubation periods for the low calcium concentration range (1 uM to

163

100 PM) and the high concentration range (250 PM to 5 mu) separately. The values are given in Table II. The two apparent Km values were around 5 PM and above 1 mM and were very similar for the analysis of the initial linear uptake rates as compared to the analysis of the curvilinear uptake rate. 'For the apparent Vmax a 5- to 7-fold lower value was obtained for,the low concentration range as compared to the high concentration range. The Vmax values based on the initial linear uptake values are considerably higher as those obtained for the prolongued incubation period; 3- to 4-fold higher for the short incubation period by comparing the values extrapolated to the same incubation time. In 2 preliminary experiments we have measured 1 set uptake values at 5 mM calcium in brush border membranes isolated from duodenum. The uptake in duodenal preparation is 1.8 to 2.4 fold higher than in jejunal preparations (data not shown) in agreement with the location of calcium transport in intact tissue.

Table

II.

APPARENT KINETIC PARAMETERS FOR CALCIUM UPTAKE AFTER DIFFERENT INCUBATION PERIODS

Data obtained in 3-5 separate experiments as in (Figure 2) were analyzed according to the Hanes-Woolf transformation ([S]/V versus [S?) of the Michaelis-Menton equation. The values were obtained in linear regression analysis with values for r' higher than 0.94. The data for the low concentration range (I&l and Vmaxl; 1 1J-Mto 100 m) and the data for the high concentration range (Km2 and Vmax2; 250 1_IM to 5 mM) were analysed separately. The values represents means f S.D. of 3-5 separate determinations.

initial linear rate (1 second incubation) (1 ~JM to

(250

uM to

(15 to

curvilinear 60 seconds

rate incubation)

100 UM)

Km1

4.98

5 1.73

)AM

5.62

-+ 1.32

v max l

0.16

+ 0.04

nmol/mg prot./sec

2.40

+ 0.036

mM

1.599

1JM nmol/mg prot./min

5 mM)

%I2

1.295

-+ 0.306

v max 2

0.83

+ 0.19

nmol/mg prot./sec

164

15.34

-+ 0.306 + 4.19

mM nmol/mg prot./min

DISCUSSION Calcium uptake into intestinal brush border vesicles and its saturation has been analyzed in brush border membrane vesicles isolated from rat mid jejunum. Similar studies have been published previously by other 10,11,28,29) and for chicken groups for mammalian preparations (e.g. A general observation in all these studies was preparations (12-18). the high equilibrium uptake for calcium exceeding that of D-glucose and suggesting extensive binding of calcium most probably to structures in the vesicle interior. A study of Rasmussen et. al. (12) suggested considerably less binding of calcium in brush border vesicles from chick intestine compared to mammalian preparations. However, recently Bikle et. al. (16) reported extensive binding within chick brush border in the studies reporting kinetic vesicle interior as well. Furthermore, constants (apparent Km and apparent V max) non-linear uptake rates were analyzed. In view of these difficulties in the previous reports we found it necessary to perform a brief study on the effect of the incubation time - initial linear uptake rate versus curvilinear uptake rate on the apparent kinetic parameters of calcium uptake by rat intestinal brush border membranes and to define whether calcium uptake measured at the two conditions fulfills some criteria of transport. It is difficult to provide clear evidence for transmembrane transport of calcium and to distinguish it from binding to the extravesicular surface. We are aware of the fact that none of the follwing observations are exclusively indicative for transmembrane transport and not for binding of calcium to the extravesicular surface. However, taken together the following arguments speak in favour of transmembrane transport of calcium as being responsible for calcium uptake at both initial linear and curvilinear uptake conditions: 1) EGTA which was present in the stop solution should have removed extravesicularly bound calcium; 2) initial linear uptake extrapolates through zero uptake for a zero incubation period, i.e. a binding to the extravesicular surface should be represented by an unusually slow process; 3) the uptake of calcium is stimulated by an inside negative membrane potential indicating electrogenic calcium flux (theoretically it could also be explained by a potential-dependent exposure of binding sites at the extravesicular surface); 4) the uptake of calcium is stimulated by increasing t$e incubftion temperature; lowering the incubation temperature from 20 C to 15 C decreases the rate by approximatively 30 % (U. Ganguli, P. Gmaj and H. Murer, unpublished observation); 5) the addition of A23187 in situations far from equilibrium stimulates calcium uptake (Table I), whereas the addition of EGTA in the presence of A23187 removes practically all calcium from the vesicles preloaded with calcium (e.g. 10, and U. Ganguli and H. Murer, unpublished observations). On the basis of these arguments we believe that the observed calcium uptake represents transmembrane transport of calcium followed by binding of calcium to sites not accessible from the extravesicular compartment. As it is clear from the present publication as well as

165

from previous publications on this topic, a study of the dependency of vesicular calcium uptake on the osmotic-sensitive space would certainly not help to define whether calcium uptake represents transmembrane transport. Due to the intravesicular binding of calcium such experiments will show binding but do not allow to distinguish between extravesicular and/or intravesicular binding (e.g. 10). The saturation experiments (Figure 2) provide evidence for at least two components in vesicular calcium uptake: at low concentrations a saturable uptake with an apparent Km of around 5 UM is observed (Table II); at high concentrations we observed a non-linear relationship of calcium uptake to calcium concentration and no saturation was obtained up to 5 mM calcium. The apparent non-saturability at the high concentrations might be related to a diffusional component superimposed to a saturable transport process although other explanations cannot be Calculation of an apparent Km for the non-saturable excluded. component suggest that the apparent affinity for this/these processe(s) is rather low, i.e. in the millimolar range (Table II). The buffered free 2+ Ca concentrations used in our experiments allow determination of calcium uptake kinetics in the micromolar range. The observed highaffinity component of 5 uM free Ca 2+ is considerably higher than the affinities reported by others in similar studies (10,ll). This difference can be most probably explained by the use of relatively high unbuffered calcium concentrations in their kinetic experiments, conceivably missing the high affinity component of calcium uptake (see Figure 2). In favour of such an explanation is the good agreement between the low affinity values for calcium uptake published by Miller and Bronner (1.1 mM; 10) and in this report (1.3-1.6 mM). It is of interest that the apparent Km values are similar for the initial linear rate and for the curvilinear rate of uptake of calcium (Table II). This suggests that at both incubation periods, the same As indicated by the data processes are determining the calcium uptake. given in Table I, calcium uptake in the absence of ionophores is far below equilibrium uptake for the 1 set as well as for the prolongued incubation period in the low and in the high calcium concentration range. In all 4 situations, the addition of the Ca-ionophore A23187 which leads to accelerated equilibration of calcium - produced a several fold increase in calcium uptake. Thus, it can be concluded that the apparent Km values observed are characteristic for a process of calcium influx into the vesicles rather than for binding to intravesicular structures. Furthermore, the decrease in the maximal uptake rate (apparent Vmax) with prolongued incubation periods seems to indicate that progressive loading of the vesicles with calcium and its binding to intravesicular structures leads to a decrease of calcium transport rate. This is an important observation, since in a recent review on intestinal calcium transport (30) it is concluded that calcium fluxes into brush border membrane vesicles are much lower than transcellular fluxes in the intact intestine.

166

The study does not allow to decide whether the multiple components (saturation experiments) are representative for a multiple step transport mechanisms mechanism in one transport process or for separate existing in the same membrane or in different membrane populations present in our preparation due to vesicle heterogeneity (e.g. 20). It is concluded that calcium uptake values obtained at incubation periods far from equilibrium uptake are characteristic for mechanisms involved Transmembrane calcium flux is potential in transmembrane calcium flux. sensitive and saturable and occurs by multiple pathways.

ACKNOWLEDGEMENTS We wish to thank Bruno Hagenbuch for providing us with a computer This work was supported by the program for the kinetical analysis. Swiss National Science Foundation, Grant No. 3.881.085 and 3.881.185. Dr. Uma Ganguli was supported by a World Health Organisation fellowship. REFERENCES 1.

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