Transport functions of the chick chorio-allantoic membrane

Experimental Cell Research 58 (1969) 107-l 17

TRANSPORT

FUNCTIONS

OF THE CHICK

CHORIO-ALLANTOIC

MEMBRANE II. Active A. R. TEREPKA,

Calcium

Transport,

in vitro

MARY E. STEWART and NANCY

MERKEL

Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, N Y 14620, USA

SUMMARY Calcium flux across the embryonic chick chorio-allantoic membrane was studied in an Ussingtype electrolyte transport chamber. Beginning at about incubation day 14, this membrane developed the ability to transport calcium actively as evidenced by net transport against large chemical gradients and under “short-circuit” conditions. Saturation kinetics were demonstrated and almost any interference with cellular energy production was deleterious to the transport process. In addition, the transport system showed specificity in that strontium was transported only one-third as well as calcium, and calcium movement was not coupled with that of anions such as phosphate and sulfate. Evidence is presented suggesting that calcium was compartmentalized or “packed” during the process of its transcellular transport. A distinction is made between normal intra-cellular calcium ion regulation and the bulk movement of calcium across cells. Tentatively, it is postulated that a very specialized and selective form of pinocytosis, or endocytosis, underlies the active trans-cellular transport of calcium by epithelial tissues.

In the preceding communication [l] it was pointed out that the endoderm of the embryonic chick chorio-allantoic (C-A) membrane was involved in active sodium transport, and this transport capability was correlated with changes in endoderm histology. Distinctive changes in the histology of the chorionic ectoderm were also noted. Ectoderma1 cell proliferation and differentiation were seen at 10 to 11 days of incubation. By the 13th day, a well demarcated and distinctive layer of cells was present, 2-4 cells in thickness, and this appearance was maintained until a few days before hatching, when atrophic and degenerative changes seemed to occur. The bulk of the calcium utilized for em-

bryonic bone formation is transferred from the egg shell to the embryo between the 13th day of incubation and hatching [2]. The chorionic ectoderm lies directly beneath the egg shell, separated from it only by the acellular shell membranes, and the calcium released from the shell must traverse these cells to gain access to the chick’s circulation. The present communication provides direct evidence that the chorio-allantoic membrane is engaged in the active transport of calcium. By adapting the morphologically simple tissue to an in vitro Ussing-type transport chamber, a clearer insight has been obtained into the nature of the trans-cellular transport of this important divalent cation. Exptl Cell Res 58

108 A. R. Terepka et al. MATERIALS

AND METHODS

Apparatus The chamber used for transport studies was essentially a modification of that first described by Ussing & Zerahn [3]. The membrane was mounted betweentwo lucite cylinders which were tongued and grooved to hold thk membrane firmly. The area of exposure was z cm2. Holes were provided in the front of the chamber into which agar-salt bridges were inserted and connected through calomel half-cells to a recorder for the continuous measurement of transmembrane potential. Carbon electrodes, t” in diameter, werecemented into the sides of the chamber and connected to a d.c. microammeter and external battery for short circuit current measurements. A glass bubble-pump assembly allowed continuous circulation and aeration of the bathing solutions (15 ml) on each side of the chamber. Small gas escape ports at the top of the bubblers allowed periodic sampling of the solutions which were introduced and removed through a T-joint at the low point of the bubblers.

Membranes Fertile White Leghorn eggs were incubated in an upright position in a commercial incubator and turned at least twice daily. An egg of approximately the desired age was first candled to ascertain that it was fertile and had a normal-appearing C-A membrane. After carefully peeling away the shell over the air space, a circular section of the C-A membrane was cut out and immediately placed on one-half of the chamber assembly. Any outer shell membrane was spirally peeled away leaving only the C-A membrane and supporting acellular inner shell membrane on the chamber. The second half of the chamber was then clamped securely over the membrane. The entire procedure required about 2-3 min of which approx. 30 set elapsed between the time the membrane was removed from its normal circulation and exposed to the pre-warmed solutions in the chamber assembly. The correct age of the embryo was determined by the staging technique of Hamilton [4].

Experimental procedure The chamber assembly with its C-A membrane was placed in an egg incubator set at 38°C (Sears Roebuck model 228-736 with trays removed) and flushed twice for 5 min with the test solution to be used in the experiment. After the last flush, fresh solution was introduced and the isotope was added. A 3 min mixing time was allowed before the first sample was take; The same 0.1 ml Lang-Levy micropipette (H. E. Pedersen Co., Copenhagen> was used to obtain repeated samples from a given side of the chamber and these were placed directly into liquid scintillation counting vials. 45Ca(1.S.A) was obtained from Nuclear Science and Engineering Corporation, Pittsburgh, Pa. The isotope was cleaned before use by oxalate precipitation (with added carrier) followed by a 2 h ashing at 500°C to Exptl Cell Res 58

convert the oxalate to carbonate. The ash was redissolved with a minimum amount of dilute HCI, evaporated to dryness and redissolved in distilled water. Approx. 50 ~1 of this stock solution (containing 34 ,uC of 45Ca) was added to the 15 ml of solution bathing one side of the membrane. All experiments were done under open-circuit conditions except where stated.

Solutions Unless indicated otherwise, the bathing solutions in both sides of the membrane had the following composition: sodium, 133 mM; potassium, 6 mM; chloride, 114 mM; bicarbonate, 25 mM; magnesium, 1.2 mM; phosphate, 1.0 mM; sulfate, 1.2 mM and glucose, 80 mg/lOO ml. The calcium concentration for the control solutions was 1.0 mM except for the special experiments described below. A 5 % CO,-95 % 0, gas mixture was used to aerate and circulate the bathing fluids and to maintain the solution pH at 7.35-7.45 throughout.

Chemical analyses At the end of each experiment, the membrane was removed from the chamber and rinsed for approx. 1 min in distilled water. The inner shell membrane and C-A membranes were detached and ashed separately in platinum crucibles at 600°C overnight. The ash was taken up in 1 ml of 3 N HCI. Of this solution, 0.1 ml was taken for 45Ca counts and 0.2 ml for total calcium analyses [5]. Another 0.25 ml was hydrolyzed in 5 ml of 0.5 N HCI for 48 h and analyzed for total phosphorus by the method of Chen, Toribara & Warner [6].

Calculations and expression of results The ectodermal surface of the C-A membrane (that facing the egg-shell proper, in vivo) is referred to in this communication as tile “outside” of the membrane, with the endodermal surface as the “inside”. In each experiment the following parameters were calculated: “Total uptake”, i.e., the total amount of calcium taken intb the membrane as measured by the disappearance of Ya counts from the solution to which the isotope was added; “trans-membrane flux” (TMF), the total amount of this calcium which appeared in the solution on the opposite side of the membrane; and “tissue uptake”, the amount of calcium transferred into the membrane based on ?a counts after ashing. The initial specific activity of the solution to which the isotope was added was used to calculate all subsequent parameters. The sum of transmembrane flux and tissue uptake, the parameters capable of being determined most accurately, is referred to as “Total transported”. Experimental results are expressed as means + standard error of the

.v

mean, I.e.,

N(N-

I)’

Chorio-allantoic

membrane

calcium transport.

II

109

RESULTS Control

experiments

A complete typical experiment using the C-A membrane from a 17 day old embryo is shown in fig. 1. The solutions bathing both sides of the membrane contained 1 mM calcium and the 45Ca was added to the outside solution. The cumulative appearance of calcium in the inside solution, the trans-membrane flux (TMF), is plotted above the abscissa in pEq/cm’ of membrane. Approx. 0.43 pEq/cm2 of calcium were transferred across the membrane in the 6 h period. The actual 45Ca counts observed in the inside solution in this experiment are indicated by the numbers above each bar. From a background count of 76 cpm/O. 1 ml sample, the count rose to 2420. The disappearance of 45Ca counts from the outside solution over the same intervals are shown below the abscissa. The initial outside solution count was 52.2 x lo3 cpm/O.l ml and this fell progressively to 47.3 x 103, a loss of 4900 counts. The decrease in counts was equivalent to 0.90 pEq/cm’ of calcium, more than twice the amount recovered in the inside solution. The difference between disappearance and appearance of calcium in the two bathing solutions was accounted for by 45Ca counts still remaining in the membrane at the end of the experiment. This is shown diagrammatically in the small bar graph on the right in fig. 1. Characteristically between 4 and i of the calcium transferred from the outside solution is recovered in the membrane. It should be noted in the figure that the disappearance of counts from the outside in the first l-2 h (Total uptake) greatly exceeded the appearance on the inside (TMF). The net transfer of calcium from the outside solution to the inside can be confirmed by chemical analysis. In the experiment shown in fig. 1, the initial bathing solutions con-

06 43

04 02

02 04 06 08

1

473

, 1

2

3

4

5

6

Abscissa: Time (hours); ordinate: cumulative flux of calcium in pEq/cm’ of membrane. Fig. I. Control experiment with 1 mM calcium on both sides of the membrane. The disappearance of calcium from the outside solution (Total uptake) is plotted below the zero base line and the appearance of calcium in the inside solution (TMF) is plotted above. The numbers above and below each bar indicate the cpm of Cad5 in the two solutions in this experiment. The small bar graph on the right represents recoveries (see text).

tained 1 mM calcium (40 pg/ml) on both sides of the membrane. After six hours, the final inside solution was 4.7 pg/ml higher than that on the outside. Transport

versus age of embryo

Using the same experimental design described above, a series of control experiments were carried out with C-A membranes from embryos varying in age from 10 to 19 days. The results are shown in fig. 2. Plotted above the abscissa is the mean TMF in pEq/cm’/h for the 6 h experiment, and the tissue uptake (based on the 45Ca counts recovered in the membrane) is plotted below. It can be seen that after day 13 there is a significantly larger transport of calcium into and across the C-A membrane. Transfer of calcium from the egg shell to the embryo for bone formation, in Exptl Cell Res 58

110

A. R. Terepka

et al.

.08 , s .06 04

lb

04 06 08 IO’

/

lo-11 12-13

14

15

16

17

18

19-20

Abscissa: Age of the embryo (days); ordinate: flux in pEq/cm’/h. Fig. 2. Transport of calcium by membranes of different embryonic age. Trans-membrane flux is plotted above and membrane uptake is plotted below the zero base line so that length of bar also represents Total transported calcium from the outside solution. Number of experiments is in parentheses within each bar and the S.E. of the mean value is shown.

vivo is known to begin at about this time [2]. Transport appears to decline after day 18, correlating with the membrane’s histological appearance [l] which showed degenerative changes at this time. The results of all the 15-19 day control experiments shown in fig. 2 are summarized in table 1. The sum of the TMF and tissue uptake (Total transported) was 0.127 pEq/ cm2/h. This agrees closely with a value of Table 1, Comparison

of forward

Solution [Ca’+] (mM)

Forward flux Back flux Back flux Exptl

Cell Res 58

0.124-L-0.005 pEq/cm2/h found for total uptake of calcium from the outside solution based upon the disappearance of 45Ca counts. Also shown in table 1 is the magnitude of the calcium backflux in 15-18 day embryo C-A membranes. With the normal 1 mM calcium solutions on both sides of the membrane, transfer of calcium from the inside to the outside solution and uptake into the membrane are very small (0.003 pEq/cm’/h). Even with a favorable concentration gradient (2 mM inside, 1 mM outside) total backflux is less than 5 % of forward flux. In vivo the chorio-allantoic endoderm is impermeable to calcium as evidenced by the progressive rise of allantoic fluid calcium coincident with water reabsorption from the allantoic sac [l]. Effect

of metabolic

inhibitors

A variety of known inhibitors of cell metabolism were tested for their effect on calcium transport with the results shown in fig. 3. When the 5 % CO, in O2 was replaced by 5 :/o CO, in N2 there was a marked decrease in both trans-membrane flux of calcium and tissue uptake. Dinitrophenol, bis-hydroxycoumarin (DicoumaroP) and oligomycin also markedly affected calcium transport. Ouabain at concentrations of 1 x 10~~ M was inhibitory, but near normal transport values were seen at 1 x 10~~M. Actinomycin D (2 x 1O-6 M) and cyclohexamide (1 x 1O-4 M) were also tested in the system and were without effect on TMF over the period studied (6 h).

flux and back flux

in normal membranes

In

out

Trans-membrane flux (pEq/cm*/h + SE.)

1.0 1.0 2.0

1.0 1.0 1.0

0.055 * 0.002 0.001 k 0.001 0.004 * 0.002

Tissue uptake

Total transported

N

0.072 + 0.004 0.002 + 0.001 0.002 + 0.001

0.127 0.003 0.006

58 10 8

Chorio-allantoic

Not shown in the figure are the results of a series of ten experiments done with 1 mM calcium at room temperature. Under these conditions total transport of calcium was 0.036 ,uEq/cm2/h, about one-quarter the value seen at 37°C (0.127 pEq/cm’/h). TMF and tissue uptake were 0.019 kO.002 and 0.017 f 0.004 pEq/cm*/h, respectively (N = 10). Backflux over the 6 h period was not significantly different for controls run at 37°C. Concentration

membrane

calcium transport.

II

111

1

04 16) 03

I 1 I51

02

I 151 I(51 002 IPI

005 ,

0125 025

0.5

IO

20 mM/I

gradients

All the experiments described above were done at a calcium concentration of 1 mM on both sides of the membrane. To evaluate the membrane’s ability to transfer calcium in the presence of significant initial concentration gradients, a series of experiments were conducted in which the concentration of calcium on the outside was altered from 0.02 to 2.0 mM while the inside solution was kept constant at 1 mM. The results are shown in fig. 4. There appears to be a direct relationship between the amount of calcium transported CONTROLSNITROGEN

DNP (2x10-~)

DICOUM. OLIGOMY OUABAIN OUABAIN IiKlo-~) (5.10-5) 11.10-51 (l&I

.06

Abscissa: mM calcium in outside solution on logarithmic scale; ordinate: flux in eEq/cm2/h. Fig. 1. Effect of alterations of outside solution calcium

concentration on calcium transport. Trans-membrane flux is above and tissue uptake below. Sum of these is equivalent to total transported calcium. Number of experiments in parentheses and mean and S.E. are plotted.

04.

and the concentration of calcium in the outside solution. The transport process seems to be saturated at about 1 mM. At outside solution concentrations of 0.05 and 0.02 mM the uphill movement of calcium was still oxygen dependent, and backflux with a 20: 1 favorable gradient (0.05 mM outside, 1 mM inside) was also very small (table 2).

02

06

Membrane Abscissa: Various experiments; ordinate:

flux in pEq/

cm’jh. Fig. 3. Effect of various inhibitors of metabolism on

calcium transport. The trans-membrane flux is plotted above zero base line, and the tissue uptake is plotted below. Number of experiments in parentheses and S.E. of the mean is shown.

calcium

and specific

activity

The total calcium and phosphorus content and dry weight of C-A and inner shell membranes were determined in eggs removed directly from the incubator. A 6 cm2 area was marked over the side and both membranes were removed, dried and ashed separately. Exptl Cell Res 58

112 A. R. Terepka et al. Table 2. Effect of nitrogen atmosphere and backjlux with large uphill [Ca’+] differences Solution [CaZ+] (mM)

out

In

0.05 0.05 0.05 0.05 0.02 0.02

1.0 1.0 1.0 1.0 1.0 1.0

Oxygen Nitrogen Nitrogen 0, Backflux Oxygen Nitrogen

Trans-membrane flux (,uEq/cm’/h x 103)

Tissue uptake

Total transported

N

9.2 + 2.6 0.6 0.5 0.1 2.8kO.5 0.1

10.0 f 0.7 1.7 1.0 0.1 1.9kO.7 0.2

19.2 2.3 1.5 0.2 4.7 0.2

5 1 1 1 5 1

As shown in the lower part of table 3, the in vivo calcium content per cm2 of 10 day old, non-transporting, C-A membranes and 18 day old C-A membranes, in vivo, was the same (0.12 f 0.02 ,umoles calcium/cm’). Phosphorus was slightly greater in the older C-A membranes. Dry weights (not shown) ranged between 1.0-1.5 mg/cm’ for both groups. The experiments shown in fig. 2 and summarized in table 1 revealed a significant membrane uptake of 45Ca in addition to the 45Ca trans-membrane flux. The total stable calcium content of these 15-19 day control membranes, exposed to 1 mM calcium solutions in vitro, is shown in the center of table 3. They contained 0.33 + 0.03 pmoles calcium/ cm2, approximately a 3-fold increase as com-

pared to their in vivo calcium content. Membrane phosphorus was not changed significantly. Inner shell membranes, in vivo, contain small amounts of calcium (0.03 fO.O1 pmoles/cm’, N = 8) and are practical1 y devoid of phosphorus. There was no significant alteration in shell membrane calcium or phosphorus, in vitro. In fig. 4 it was seen that the total transport of calcium was directly related to the concentration of calcium in the outside solution. Maximal transport values were seen at about 1 mM. The calcium and phosphorus content of the ashed membranes from these experiments are also shown in table 3. With outside solution calcium concentrations less than 1 mM, there was less calcium present in the C-A

Table 3. Effect of outside [Ca’+] on membrane calcium, phosphorus and specific activity Chorio-allantoic membrane Outside solution, CaZ+ concentration h-M

Calcium (pmoles/cm’)

Phosphorus (flmoles/cm’)

S.A. ratio (Memb./out. soln.)

N

In vitro 0.02 0.05 0.10 0.50 1.OO(Control) 1.50 2.00

0.10+0.01 0.15+0.03 0.15&0.02 0.19*0.03 0.33 * 0.03 0.32 f 0.03 0.30+ 0.03

0.64 + 0.09 0.66? 0.03 0.59 f 0.05 0.52 & 0.02 0.63 + 0.03 0.55 + 0.06 0.69 + 0.07

0.04 f 0.01 0.28+0.10 0.33 + 0.07 0.75 + 0.04 0.66 + 0.03 0.63kO.14 0.69 + 0.07

5 5 10 5 15 5 6

In vitro IO-day embryos 18-day embryos

0.12+0.02 0.12*0.01

0.46 + 0.06 0.54*0.04

Exptl Cell Res 58

5 4

Chorio-allantoic membranes at the end of the experiments. Above 1 mM there was no further increase in tissue calcium. Membrane phosphorus, on the other hand, was not altered significantly in any of these experiments, The last column in the table shows the calcium specific activity of the membranes in the various experiments compared as a ratio with the calcium specific activity of the outside bathing solution. Even after 6 h of exposure to 45Ca the membrane specific activities were always less than that of the outside solution and decreased roughly in proportion to the solution calcium ion concentration. An additional series of experiments were done in 1 mM calcium with 45Ca on both sides of the membrane. The specific activity of the C-A membrane was determined after 6 h and compared with that of the bathing solution. The results were similar; i.e., membrane/ solution specific activity ratios were consistently less than unity (0.74kO.03, N = 10). Specific activity of transported calcium It was pointed out above that by chemical titrations, a net increase of calcium could be detected in the inside solution and this was associated with a decrease in outside solution calcium concentration. Since membrane specific activities never reached the specific activity of the outside bathing solution, a question of theoretical significance is the specific activity of the calcium actually being transferred into the inside solution. In an attempt to evaluate this, a series of experiments were conducted using 0.1 mM calcium as the bathing solution. As seen in table 3, under these conditions membrane specific activities were only about one-third that of the outside solution. Samples in triplicate were taken of the inside solution at 3 min and at 6 h and analyzed for total calcium and 45Ca. From the net increase in stable calcium and the radioactivity accumulated, the specis-

691807

membrane calcium transport. II

113

fit activity of transported calcium was calculated and compared with that of the corresponding outside solution, In fifteen experiments done in this way, the mean increase in inside solution calcium was 0.65 pg/ml. The mean 45Ca increase was approx. 58,000 cpm/ml. Comparing individually the inside specific activity with that on the outside, a mean ratio of 0.80+0.05 (N = 15) was obtained which is significantly different (p
114 A. R. Terepka et al. Table 4. Transport of strontium, phosphate and sulfate and the effect of stripping Trans-membrane flux

WaO YSr” 32PO4 T30, Stripped ControV

(,uEq/cm’/h + s.E.)

Tissue uptake

Total transported

N

0.043 + 0.006 0.012+0.001 0.004 + 0.001 0.002 + 0.001 0.005 + 0.002 0.055 + 0.002

0.053 i- 0.006 0.018 + 0.002 0.024+ 0.009 0.002 + 0.002 0.002 + 0.001 0.072? 0.004

0.096 0.030 0.028 0.004 0.007 0.127

s8 7 6 4 58

a Solutions contained 0.5 mM calcium and 0.5 mM strontium. b Normal controls (fig. 2, table 1) with 1.0 mM calcium solutions.

bathing solutions contained 0.5 mM calcium chloride and 0.5 mM strontium chloride. In eight experiments 45Ca was added to the outside solution; another eight had 89Sr as the tracer. The results are shown in table 4. Trans-membrane flux and tissue uptake of strontium was about one-third of that seen for calcium. The transfer of 32P-labeled phosphate and 35S-labeled sulfate into and across the C-A membrane was also determined. The regular Krebs-Ringer-Bicarbonate solution contained both these anions (1 mM P04; 1.2 mM SO,). As shown in table 4, trans-membrane flux for phosphate was less than l/lOth the normal calcium TMF values shown in the bottom line of the table. Tissue uptake of phosphate based on 32P counts was significant (0.024 pEq/cm2/h) but only about one-third that seen for calcium (0.072 pEq/cm2/h). Since total membrane phosphorus is not changed significantly, this probably represents isotope exchange. Sulfate TMF and uptake into the membrane were very low. Membrane potential The C-A membrane in vitro develops a transmembrane potential. Under the experimental conditions employed, we found this to be variable and unrelated to the calcium fluxes Exptl

Cell Res 58

described above. Consistent trans-membrane potentials were observed, however, with Trisbuffered solutions and these were approx. + 5.0 mV (outside positive relative to inside). This potential gradually approached zero toward the end of the 6 h experiment. Shortcircuit current measurements were in the order of 30-40 PA/~ cm2 with a membrane potential difference of + 5.0 mV and declined in concert with the gradual decline in membranepotential. Although we consider the electrical characteristics of the membrane unrelated to calcium transport (a detailed investigation of this aspect of C-A membrane function has been completed [7] and will be reported elsewhere) we did determine the effect of continuous short-circuit procedures on calcium transport. There was no statistical difference in total calcium transport in the normal membranes as compared with those continually short-circuited. Effect of stripping In the routine preparation of the C-A membranes for in vitro transport studies, the membrane was mounted in the chamber assembly with the inner shell (I-S) membrane from the air space still attached. It was felt that the acellular I-S membrane served only

Chorio-allantoic as a support for the underlying C-A membrane during the preparative manipulations. The two membranes after 14 days of incubation are firmly attached to each other, and it soon became apparent that the integrity of this junction was crucial for the active transport process to occur. If the I-S membrane is carefully stripped off the C-A membrane so as not to produce any tears, both calcium TMF and tissue uptake are dramatically reduced (table 4) when compared with normal control values for TMF and tissue uptake. Backflux in these stripped membranes was not significantly different from forward flux so that there is no net transfer of calcium. The total calcium content of the ashed membranes from these experiments was 0.05 + 0.02 ~moles/cmz, about half that of the in vivo value 0.12 pmoles/cm2 (table 3). DISCUSSION In most of the higher animal species, no substance is under more rigorous homeostatic control than is serum and extracellular fluid calcium [8]. To accomplish this, a delicately balanced system of transport functions has evolved integrating absorption from the gut with reabsorption from the kidney, and with flow to and from the skeleton [9]. In each case, the trans-membrane transfer of calcium poses a threat to the metabolic well-being of the very cells engaged in the process, since high levels of intracellular calcium can, for example, uncouple oxidative phosphorylation in mitochondria [lo] or markedly inhibit pyruvate kinase [I I] and pyruvate carboxylase activity [12]. For these reasons it is generally accepted that the intracellular levels of calcium are maintained at very low levels, of the order of 1O-6 M [13]. While few direct determinations of intracellular calcium have been made in tissues other than muscle [14], virtu-

membrane calcium transport. II

1I5

061 FLUSH OUT

04 02

02 04 06 08

1

2

3

4

5

Abscissn~ Time (hours); ordinate:

membrane.

6 7 flux in pEq/cm2 of

Fig. 5. Flush-out experiment after 34 h of mem-

brane exposure to ‘Wa. Cumulative disappearance of calcium from outside solution is plotted below zero base line. Trans-membrane flux is plotted above.

ally all of the calcium present in most whole tissues can be attributed to the extracellular compartment [ 151. Apparently, the intracellular regulation of calcium ion levels involves a “pump-leak” mechanism, but details of how this is accomplished remain to be settled. Wallach, Reizenstein & Bellavia [16], studying liver slices concluded that the influx of calcium is not passive. In contrast, Borle [17] recently suggested that the calcium influx is passive and the efflux involves an active, metabolically supported pump. In any case, those cells engaged in the vital function of trans-cellular transport of calcium are faced with an additional and special problem. How can bulk transfer be accomplished without raising the intracellular level of ionic calcium above lo-’ M? The chick chorio-allantoic membrane is an epithelial tissue called upon to transfer large quantities of egg-shell calcium to the embryo at a certain stage in its development. This tissue appears to have solved the problem by compartmentalizing the calcium in transit, thus preventing the transport system from disExptl Cell Res 58

116 A. R. Terepka et al. rupting the internal machinery of the cell. Perhaps this transport system is unique to the avian egg. On the other hand, in view of the biochemical similarity of all living systems, it is possible that the C-A membrane is unique only in that it represents the one tissue that lends itself to convenient study of transmembrane transport of calcium in the laboratory under in vitro conditions. Morphologically, at least, it is the simplest tissue currently available for transport chamber studies of calcium movements. The present investigation provides conclusive evidence that the C-A membrane transports calcium actively. Transport occurs against large chemical gradients and under “short-circuit” conditions. The kinetics show saturation of the mechanism at high concentration of calcium in the outside solution. Almost any interference with energy production is deleterious to the transport process. Decreased temperature, uncouplers of oxidative phosphorylation (such as dinitrophenol and Dicoumarol@), oligomycin and lack of oxygen are all strongly inhibitory. From these data, it is clear that the process is “active” and requires the support of oxidative phosphorylation. In addition, the system shows considerable specificity. Strontium is transported only a third as well as calcium. Moreover the movement of calcium is not coupled with that of anions such as phosphate or sulfate. All of these observations would be compatible with a transport system analogous to that believed to move sodium and potassium ions [18]. However, all of the remaining data suggest that the transport of calcium is quite different. First of all, the transport system seems to be non-electrogenic. No correlation between transport of calcium and the transmembrane potential was observed and voltage clamping in either direction had little affect on the process [7]. Secondly, the kinetics are uniExp/l

Cell Res 58

que. There is first an uptake by the membrane, and following a lag period, labelled calcium appears in the inside solution. When the labelled outside solution is replaced with an unlabelled calcium solution, the “pulse” present in the membrane continues on through to the opposite side with little backflow. Finally, the flow of labelled calcium does not appear to mix homogeneously with the calcium in the membrane itself. It was emphasized that even after 6 h exposure to 45Ca the specific activity of calcium in the membrane was consistently less than the specific activity of the outside bathing solution (table 3). The data obtained for the specific activity of the calcium transported into the inside solution suggest that this is significantly higher than the specific activity of the membrane itself and close to that of the outside solution. Such data are consistent with the concept of compartmentalization of the calcium undergoing active transport but do not define the nature of the mechanism. However, electron microscopic studies of the C-A membrane in our laboratory suggest that the cells lying between the inner shell membrane and the respiratory capillaries are actively engaged in pinocytosis. Other than the calcium transport reported here, no other transport function has been ascribed to the chorionic ectoderm. Since these cells are joined by “tight junctions” and material cannot flow around them [19], it is tempting to assume that calcium in transport is somehow compartmentalized in pinocytotic vesicles before gaining access to the embryonic circulation. Some support for this assumption was recently obtained with the use of the electronmicroprobe. In the transporting layer of cells, calcium was not uniformly distributed and discrete “packages” of calcium were identified [20]. It is of some interest that the three mammalian tissues involved in calcium transport,

Chorio-allantoic intestinal epithelium, kidney tubules and osteoclasts of bone [21, 221 have all been shown to be engaged in active pinocytosis. It will be experimentally difficult to establish whether this interesting correlation, at present only circumstantial, is truly indicative that a single mechanism for the trans-membrane transport of calcium (i.e., a very selective form of pinocytosis or endocytosis) is common to all these tissues. Dr William F. Neuman offered valuable advice and constant encouragement. Technical assistance was provided by Constance Curran, Betty Risen and Martha Slocumb. A.R.T. is recipient of a USPHS Career Development Award, 9-K3-AM-7876. This work was supported in part by USPHS grants 5 ROI AM 08271 and 1 Tl DE 175 and in part under contract with the USAEC at the University of Rochester Atomic Energy Project and has been assigned Report No. UR-49-1085.

REFERENCES I. Stewart, M E & Terepka, A R, Exptl cell res 58 (1969) 93. 2. Johnston, P M & Comar, C L, Am j physiol 183 (1955) 365. 3. Ussing, H H & Zerahn, K, Acta physiol stand 23 (1951) 110. 4. Hamilton, H L, Lillie’s development of the chick. Holt, Rinehart & Wilson (1952). 5. Toribara, T Y & Koval, L, Talanta 7 (1961) 248.

membrane calcium transport. II

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