J. Cell Sd. 82, 85-97 (1986)
85
Printed in Great Britain © The Company of Biologists Limited 1986
CALCIUM-TRANSPORT FUNCTION OF THE CHICK
EMBRYONIC CHORIOALLANTOIC MEMBRANE
II. FUNCTIONAL
INVOLVEMENT OF CALCIUM-BINDING
PROTEIN, Ca2 + -ATPase AND CARBONIC ANHYDRASE
ROCKY S. TUAN*, MONICA J. CARSON, JUDITH A. JOZEFIAK,
KATHY A. KNOWLES AND BARBARA A. SHOTWELL
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
SUMMARY
This study aimed to investigate the mechanism of active calcium transport in the chick
embryonic chonoallantoic membrane (CAM) by assessing the functional involvement of three
previously identified, putative components of the transport pathway. These components are a
calcium-binding protein (CaBP), Caz+-activated ATPase and carbonic anhydrase. Using specific
reagents, including antibodies and enzyme inhibitors in vivo and in vitro in CAM calcium uptake
assays, it was shown that these biochemically identified components were all functionally involved.
The results of these studies also indicate that active calcium uptake by the CAM requires the
presence of the CaBP on the cell surface in a laterally mobile manner, while carbonic anhydrase
appeared to be a cytosolic component. We further analysed the subcellular location of the calciumuptake activity by gel filtration and density-gradient fractionation of cell-free microsomes of the
CAM and the results suggest that this activity is associated with the plasma membrane.
INTRODUCTION
Calcium translocation by the chick embryonic chonoallantoic membrane (CAM)
is a developmentally regulated, uni-directional and calcium-specific active-transport
function (Garrison & Terepka, 1972a,b; Terepka et al. 1976; Tuan & Zrike, 1978;
Dunn, Graves & Fitzharris, 1981; also see accompanying paper). Previous studies
carried out in this laboratory have led to the identification of three biochemical,
macromolecular components that appear to play functional roles in CAM calcium
transport. These are: (1) a specific, high-molecular-weight calcium-binding protein
(CaBP) (Tuan & Scott, 1977; Tuan et al. 1978a,6,c; Tuan, 1980a); (2) a Ca 2+ activated, Mg2+-dependent ATPase (Tuan & Knowles, 1984); and (3) carbonic
anhydrase (Tuan & Zrike, 1978; Tuan, 1984). All three proteins are localized in the
ectoderm of the CAM, which lies adjacent to the shell membrane and eggshell (the
calcium source), and constitutes the calcium-transporting cell layer of the CAM
(Coleman & Terepka, 19726). Specifically, the CaBP is associated with the cell
surface of the ectoderm (Tuan et al. 19786); the Ca2+-ATPase is an integral
membrane component, which appears to be a near neighbour of the CaBP (Tuan &
Knowles, 1984), and the carbonic anhydrase is most probably a cytosolic component
• Author for correspondence.
Key words: calcium transport, subcellular fractionation, plasma membrane, endocytosis.
86
R. S. Tuan and others
(Anderson et al. 1981; Tuan, 1984). The involvement of these components in CAM
calcium transport is suggested by a close temporal and spatial correlation between
their appearance and the onset of CAM calcium transport in the developing chick
embryo.
To define the roles of these putative transport components requires: (1) the
availability of specific reagents for targeted perturbations of these components in situ
in the CAM, and (2) calcium-transport assay systems that would permit the
application of the aforementioned reagents for directly evaluating the functional
importance of these components. Reagents specific for each of these components are
indeed available. These include specific antibodies raised against the CaBP (Tuan et
al. 19786), pharmacochemical inhibitors, such as quercetin, for the CAM Ca 2+ ATPase (Tuan & Knowles, 1984) and sulphonamides, such as acetazolamide, for the
CAM carbonic anhydrase (Tuan & Zrike, 1978; Tuan, 1984). Using these reagents
in conjunction with the two in vitro calcium-uptake assay systems (CAM tissue disks
and cell-free microsomal membranes) discussed in the preceding paper and with an
in vivo system (Tuan & Zrike, 1978), we directly tested the functional involvement
of the CaBP, the Ca2+-ATPase and carbonic anhydrase in CAM calcium transport.
Results reported here strongly indicate that these components are integrally and
functionally involved in CAM calcium transport. These results also provide further
insight into the mechanism of the transport function and have revealed its association
with the plasma membrane.
MATERIALS AND METHODS
Chick embryos and CAM
Fertilized white Leghorn chicken eggs were incubated at 37-5 °C in a humidified commercial eggincubator for the desired period of time. Whole CAM was harvested by dissecting it away from the
embryo and was rinsed clear of adhering materials with cold physiological saline.
Preparation of microsomes
This was carried out as described in the accompanying paper. Microsomes were suspended in a
buffer containing lOmM-imidazole, pH7-0, containing 0-1 M-KC1 (buffer C) until use.
Fractionation of microsomes
Two methods were used to fractionate whole CAM microsomes. (1) Gel filtration. Size
fractionation of microsomal membranes was carried out on a Sephacryl S-1000 (Pharmacia
Biochemicals) column (1 cmX28cm) eluted with 0*6M-KC1, lOmM-imidazole, pH7-4, containing
0-3M-sucrose at a flow rate of 32nilh" 1 . The sample load was routinely 1-2ml. (2) Densitygradient centrifugation. Microsomes in buffer C were made up to 10% sucrose (w/w) and 1-5 ml
was loaded on top of a step sucrose gradient (20 % to 55 % in 5 % increments of 1 -44 ml each). After
centrifugation in a Beckman SW41 rotor at 20 500 rev. min" 1 for 3h at 4°C, the gradient was
fractionated by upward elution into nine equivolume fractions using a gradient fractionator (ISCO
Inc.). All fractions were suspended in buffer C and collected by centrifugation at 80000£ for
80min and were suspended in buffer C until use.
Assay of CAM calcium uptake
Calcium uptake in situ. The procedure used has been described (Crooks & Simkiss, 1975; Tuan
& Zrike, 1978; Tuan, 19806, 1983; preceding paper). Calcium uptake activities at 25°C were
expressed as mol calcium min~' cm" 2 .
Calcium transport by chorioallantoic membrane. II
87
Calcium uptake by tissue disks in vitro. The procedure was as described in the accompanying
paper. Calcium uptake activities were expressed as mol calcium min~' cm" 2 .
Calcium uptake by microsomal and subcellular membranes in vitro. The procedure was similar
to that recently used for human placenta] microsomes (Tuan, 1985) and is described in the
accompanying paper. Activities were calculated as mol calcium s~' mg protein" 1 .
Enzyme assays
The procedures for the following enzyme assays all involved the measurement of released
inorganic phosphate, which was determined using the Malachite Green method as described
previously (Tuan & Knowles, 1984). All assays were carried out at 37 °C using preparations
solubilized in 1 % Triton and activities were expressed as mol phosphate released min" 1 .
Ca2+-activated ATPase. This was carried out as described previously (Tuan & Knowles, 1984)
using 1 mM-ATP, and activity was defined as the difference between ATPase activity levels
measured in the absence of Ca (i.e. 2 mM-EGTA added to the assay mixture) and in the presence
of additional 10mM-CaCl2.
Na+, K*-ATPase. The assay was carried out essentially as described previously (Tuan, 1979),
in SOmM-Tris, pH8-0, containing 3 mM-ATP, (H12M-NaCl, 20mM-KCl (2 mM-EGTA was
included to eliminate Ca 2+ -ATPase activity). The activity was defined as the difference between
ATPase activity levels measured in the absence and in the presence of 1 mM-ouabain.
5'-Mononucleotidase. The assay mixture contained 20 mM-AMP as described previously (Tuan,
1979).
Glucose-6-phosphatase. The assay mixture contained 80 mM-glucose 6-phosphate in a histidine
buffer, pH6-5, as described previously (Tuan, 1979).
Carbonic anhydrase activity was assayed on the basis of the hydration of CO2 as described
previously (Tuan & Zrike, 1978).
CaBP assay
Rabbit-derived, specific anti-CaBP antisera (Tuan et al. 19786) were used to determine CaBP
levels by means of the enzyme-linked immunosorbent assay (ELISA) (Hudson & Hay, 1980).
Briefly, samples to be tested were serially diluted and adsorbed onto the wells of a polystyrene
microtitre plate (96-well; Linbro, Hamden, CT) in a carbonate buffer, pH9-0. After washing,
specific anti-CaBP antiserum (1/250 dilution) was applied to the wells. After incubation for 2h at
room temperature, the plate was washed and the enzyme-linked secondary antiserum (alkaline
phosphate-conjugated goat anti-rabbit immunoglobulin G (IgG), Sigma Chemical Co., 1/1000
dilution) was added. After additional incubation for 2h and further washing, the ELISA was
developed usingp-nitrophenyl phosphate (Hudson & Hay, 1980) as a substrate. All samples were
compared with a stock standard consisting of an extract of CAM prepared from 18-day chick
embryos.
Preparation of anti-CaBP IgG and Fab'
Fractionation of IgG from anti-CaBP antisera was carried out as previously described (Tuan et
al. 19786). Monovalent Fab' fragments of the IgG were obtained by pepsinization followed by
reduction and alkylation and fractionation on a Sephadex G-100 (Pharmacia) column according to
standard protocols (Stanworth & Turner, 1978).
Protein assay
Protein was determined by the method of Lowry et al. (1951) with bovine serum albumin
(Sigma Chemicals) as a standard.
Electron microscopy
Tissue samples of CAM were processed for electron microscopy as described previously (Tuan &
Chang, 1975): fixation with 2-5 % glutaraldehyde in cacodylate buffer (pH7-4), postfixation with
88
R. S. Tuan and others
osmium tetroxide, and embedding in Epon. Ultrathin sections were stained with lead and uranyl
acetate, and examined on a JEOL 100S electron microscope.
Reagents
All chemicals used were of reagent grade. Radiolabelled compounds were purchased from
Amersham Corp. (Chicago, IL). Sources for the following compounds were: quercetin, ethacrynic
acid, p-nitrophenyl phosphate, ATP (vanadate-free), and tetracaine (Sigma Chemicals); trifluoperazine (Boehringer-Mannheim Biochemicals); and acetazolamide (Diamox, Lederle Labs).
Goat-derived anti-(rabbit IgG) antibodies were obtained from Miles Laboratories.
RESULTS
Involvement of CaBP, Ca2+-ATPase and carbonic anhydrase in CAM calcium
uptake
CaBP. The availability of specific anti-CaBP antibodies (Tuan et al. 19786;
Tuan, 19806) made it possible to study directly the functional involvement of the
CaBP by observing the effect of these antibodies on CAM calcium uptake. This was
first studied using the in vivo method, which involved the construction of an uptake
chamber on top of the CAM in ovo (Crooks & Simkiss, 1975; Tuan & Zrike, 1978).
With this system, we observed that pre-incubation of CAM with anti-CaBP IgG
resulted in substantial reduction of uptake activity in a dose-dependent manner,
which resembled a standard immunotitration profile (Fig. 1A). On the other hand, a
similar treatment with pre-immune serum resulted in no inhibition. The finding that
an 'immunoequivalent point' existed in the antibody-mediated inhibition of uptake
suggested that immobilization due to immuno-crosslinking could be the mechanism
of inhibition and that, furthermore, CaBP existing in a 'mobile' state on the CAM cell
surface was a requirement for functional CAM calcium uptake. This supposition was
further supported by the finding that monovalent Fab' fragments derived from antiCaBP antibodies exhibited no inhibitory effect on CAM calcium uptake (Fig. 1A).
In addition, it was found that inhibition could also be achieved in excess anti-CaBP
(1/100 dilution) if a secondary antibody (goat-derived anti-rabbit IgG) was applied
in tandem (activities: control, 100%; anti-CaBP alone, 50%; goat anti-rabbit IgG
alone, 98%; anti-CaBP plus anti-rabbit IgG, 28%). This finding was therefore
consistent with the supposition stated above. The inhibitory effect of anti-CaBP on
calcium uptake was also observed using the tissue disk method in vitro (Fig. IB). On
the other hand, calcium uptake by CAM microsomes was only slightly affected when
they were pre-treated with anti-CaBP antibodies (Fig. IB). Since CAM microsomes
contained CaBP (see below), the failure of the anti-CaBP antibodies to inhibit
calcium uptake significantly could be due to the inaccessibility of CaBP to the
antibodies, perhaps because CaBP was segregated within the relatively intact
membranous vesicles (see Fig. 2A).
Ca2+-ATPase. We have previously identified several pharmacochemical inhibitors
of the CAM Caz+-ATPase (Tuan & Knowles, 1984). When tested in the microsomal
calcium-uptake assay, both quercetin and tetracaine were effective inhibitors of
100
^
A
u
\
60
§•
E
•a 40
•3
Pi
4
N:
V
20
1
2
89
c
3
4
Anti-CaBP IgG (dilution log10)
100
2
60
en
s.
3
E
•0 40
•u
20
06
Anti-CaBP IgG (dilution log10)
Fig. 1. Effect of anti-CaBP antibodies on CAM calcium-uptake activity. A. In vivo CAM
calcium uptake assayed in 17-day and 18-day embryos as described previously (Tuan &
Zrike, 1978). B. In vitro calcium uptake using microsomes ( • — - # ) or tissue disks
(P
O) isolated from 17-day and 18-day embryos. The anti-CaBP antibodies were
IgG fractions isolated as described in Materials and Methods and reconstituted to the
original volume of the serum. All dilutions of the antibodies were made in the respective
uptake assay buffers. In A, the effect of pre-immune serum (O) and monovalent Fab
fragments of anti-CaBP IgG (A) on in vivo calcium uptake are also presented. In B,
microsomes were pre-incubated with anti-CaBP antibodies at the indicated dilutions for
30 min at 4°C with shaking before measurements of calcium-uptake activity; tissue disks,
on the other hand, were exposed to antibodies only at the time of uptake assay. All
activities (results of 3-5 separate experiments) were expressed as percentages (±s.E.) of
that in controls that contained no anti-CaBP antibodies.
R. S. Tuan and others
90
CAM calcium uptake (Table 1). On the other hand, ethacrynic acid elicited only
limited inhibition of uptake (Table 1). Since these compounds, which are effective
inhibitors of detergent-solubilized CAM Ca2+-ATPase (Tuan & Knowles, 1984),
differ in their solubilities in lipids, the above findings could have resulted from
differences in membrane partitioning efficiency. It was also previously observed
(Tuan & Knowles, 1984) that anti-calmodulin agents, such as phenothiazine, did not
inhibit CAM Ca2+-ATPase activity. As assayed here, trifluoperazine, a potent anticalmodulin phenothiazine (Cheung, 1982), also did not effectively inhibit CAM
microsomal calcium uptake (Table 1). Finally, quercetin and ethacrynic acid were
Caldum uptake
illlli,ii.,
CaBP
I 0
3 1
Pi
1 llhillh
Ca 2+ -ATPaie
1
0
l
GIUCOK-6-
llllln.l
•
ll 1,. 1
I
0-4
0-3
l
l
1
1\
! \
/
0-2
01
A
/ \
•-*
\
i
i
—
10
J
20
30
Fraction number
40
50
Fig.
2. Fractionation of CAM microsomes by gel filtration. Chromatographic fractionation of CAM microsomal membranes (17-day embryos) was carried out on a
Sephacryl S-1000 column as described in Materials and Methods and the fractions were
assayed for activities of calcium uptake, CaBP, Ca z+ -ATPase, and glucose-6-phosphatase
(see Materials and Methods for assay protocols). All activities (activity/fraction) were
expressed relative to the highest level in the chromatogram.
Calcium transport by chorioallantoic membrane. II
91
Table 1. Effect of various agents on CAM calcium uptake activity
Relative calcium uptake activity (%)*
C A M tissue
disks
Treatment
100
Control
Ethacrynic acidf
Quercetinf
Tetracainef
0-01
0-1
1
10
0-01
0-1
0-2
1
0-1
1
10
Trifluoperazinef
1
10
Acetazolamidef
10
20
SO
CAM
microsomes
100
59 ± 8 (2)
99 ± 9 (2)
85 ± 20 (2)
46 ± 1 6 (2)
—
101 ± 3 ( 1 )
87 ± 3 ( 1 )
84 ± 6 (2)
102±3(l)
50 ± 3 (2)
50 ± 2 (2)
44 ± 17 (2)
54 ± 8 (2)
32 ± 6 (2)
38 ± 4 (2)
82 ± 5 (2)
87 ± 7 (2)
49 ± 9 (2)
43 ± 8 (2)
112±6(3)
89 ± 1 4 (2)
88 ± 15 (2)
• Calcium uptake activities were assayed on the basis of kinetic measurements as described in
Materials and Methods of the accompanying paper and are expressed as percentages (±S.E.)
of control values. In these measurements, triplicate samples were used in CAM tissue-disk
experiments for all time points and quadruplicates were used for the microsomal experiments. The
number of experiments, each using ~20 embryos (day 16—17), for the data points is indicated
in parenthesis. The ranges of control values of calcium uptake in these experiments were:
40-50 pmolmin" 1 cm" 2 (tissue disks) and 8—14pmols~'mgprotein" 1 (microsomes).
f Samples were pre-incubated for 15-30min (25°C for tissue disks, 4°C for microsomes) in
uptake buffer containing the agents (concn in /ZM) at the indicated concentrations immediately
before assay and were compared with controls incubated similarly in the absence of the agents.
also tested in the tissue disk calcium-uptake assay (Table 1), which showed that both
effectively inhibited calcium uptake, with the latter requiring a substantial period of
pre-incubation of the tissue with the inhibitor. Taken together, these findings are
therefore consistent with and strongly suggest the functional participation of the
Caz+-ATPase in CAM calcium uptake.
Carbonic anhydrase. Consistent with our previous observation that treatment of
intact CAM in situ with sulphonamides (specific carbonic anhydrase inhibitors)
strongly inhibited calcium uptake (Tuan & Zrike, 1978), we found that the
sulphonamide, acetazolamide, was also a potent inhibitor of calcium uptake by CAM
tissue disks in vitro (Table 1). However, when tested in the cell-free microsomal
system, acetazolamide was an ineffective inhibitor (Table 1). Furthermore, no
detectable carbonic anhydrase activity was present in the microsomal preparations.
The involvement of carbonic anhydrase was also tested using an antiserum specific
for chick CAM carbonic anhydrase (Tuan, 1984). It was found that the antiserum
92
R. S. Tuan and others
did not inhibit calcium uptake in either the tissue disks or the microsomal system
(data not shown). These findings are therefore consistent with carbonic anhydrase
being a cytosolic component that is probably involved as a facilitative rather than an
intrinsic component of transmembrane calcium uptake (see Discussion).
Fractionation of calcium-uptake-competent membrane vesicles
To examine further the functional association between the CaBP and the Ca + ATPase and CAM calcium-uptake activity, and to gain insight into the subcellular
membrane location of uptake activity, the CAM microsomal preparations were
fractionated on the basis of size (gel filtration, Fig. 2) and density (sucrose densitygradient centrifugation, Fig. 3).
Upon gel filtration on Sephacryl S-1000, CAM microsomes were separated into
fractions according to their size, with most of the vesicular components (O'l—0-3 /zm
in diameter) probably eluting at the column void volume (Dickson et al. 1983;
Reynolds et al. 1983). Other fractions (Fig. 2) that eluted in the resolved and
salt volumes probably contained small vesicles and membrane sheets, and nonspecifically adsorbed proteins. The chromatogram was then analysed with respect to
calcium-uptake activity, enzyme activities and CaBP levels. The results in Fig. 2
clearly showed that CaBP, Ca2+-ATPase and calcium-uptake activities were largely
coincident at the column void volume and that their distribution profiles differed
considerably from that of glucose-6-phosphatase, a marker enzyme for endoplasmic
reticulum (de Duve et al. 1962).
A similar distribution was also observed upon density-gradient fractionation of
the microsomal membranes. Specifically, as shown in Fig. 3, co-distribution was
observed for Ca -ATPase activity, CaBP, calcium-uptake activity, and activities of
a plasma membrane marker enzyme, 5'-mononucleotidase (de Duve et al. 1962), all
of which differed substantially in distribution compared with glucose-6-phosphatase
(endoplasmic reticulum marker; de Duve et al. 1962) and acid phosphatase
(lysosomal marker; de Duve et al. 1962;data not shown).
These findings are therefore consistent with the notion that the CaBP and the
Ca2+-ATPase are associated with, and are probably functional components of, the
CAM calcium-uptake mechanism. In addition, the enrichment of uptake activity,
CaBP and Ca2+-ATPase in fractions that were also enriched in marker enzymes of
the plasma membrane strongly suggests that the CAM calcium-uptake system is most
probably a component of the plasma membrane.
DISCUSSION
Our results demonstrate the functional involvement of the previously identified,
putative transport components: the CaBP, the Ca +-ATPase and carbonic anhydrase
in calcium uptake by the chick embryonic CAM. Furthermore, these data strongly
indicate that the calcium-uptake activity is associated with the plasma membrane.
Although the exact modes of action of these components remain to be elucidated, the
Calcium transport by chorioallantoic membrane. II
93
findings reported here, taken together with previous observations (Terepka et al.
1976; Coleman & Terepka, I972a,b; Garrison & Terepka, \97Za,b; Crooks &
Simkiss, 1975; Anderson et al. 1981; Dunn et al. 1981; Tuan, 1980a,6, 1983, 1984;
Tuan & Knowles, 1984; Tuan & Scott, 1977; Tuan & Zrike, 1978; Tuan et al.
I978a,b,c), allow certain speculations.
We have previously postulated (Tuan & Zrike, 1978; Tuan, 1984) that carbonic
anhydrase may provide localized acidification in the ectodermal zone to promote in
vivo the dissolution of the eggshell calcite (CaCO3) mineral to produce ionized
calcium ready for uptake and, or, to regulate the metabolic fate of the HCC>3~
released from the shell. Since shell dissolution was not necessary in calcium uptake
measured in vitro as described here, our findings suggest that the enzyme is perhaps
10
Cakium uptake
CaBP
1 o
|
1 2-0
Ca2+-AT1
2 0-5
& 2
5'-monon uclec tidu K
0
2
0
2-5
|
|
|
|
Gluctue-6-pha ipha u c
| 1
1-20
1-15
1-5
110
00
E
1-05
o
1 2 3 4 5 6 7 8 9
Fraction number
Fig. 3. Fractionation of CAM microsomes of 17-day embryos by density-gradient
centrifugation. This was carried out on a sucrose density gradient (20 % to 55 %, w/w) as
described in Materials and Methods. All fractions were assayed for activities of calcium
uptake, CaBP, Ca z+ -ATPase, 5'mononucleotidase and glucose-6-phosphatase. The
chromatogram represents specific activities expressed relative to that in the original
microsomes.
94
R. S. Tuan and others
involved in specific, regional acidification to increase localized ionization or the
accessibility of transport component(s). Moreover, the fact that active calcium
uptake may take place in a subcellular membrane preparation containing no carbonic
anhydrase activity strongly suggests that the enzyme is most probably not an integral
part of the active transport mechanism, but instead participates in a facilitative role.
The functional involvement of the CaBP in CAM calcium transport has been
suggested previously from many lines of experimental evidence (Tuan, 1980a,6,
1983; Tuan & Knowles, 1984; Tuan & Scott, 1977; Tuan etal. \978a,b,c). Further
indirect evidence is also reported in the accompanying paper, i.e. the almost identical
ion specificity and affinity of CaBP and calcium-uptake activities. More importantly,
the inhibition of calcium uptake by CAM tissue (in vivo and in vitro) by exogenously
applied anti-CaBP antibodies reported here (Fig. 1) is the first direct demonstration
of the functional necessity of the CaBP in CAM calcium transport. The inability of
the anti-CaBP antibodies to inhibit microsomal calcium uptake indicates that the
subcellular vesicles are probably inside-out structures, with the CaBP located inside
and therefore inaccessible to the exogenously applied antibodies. In this manner, the
CaBP therefore serves as an internal calcium sink for the subcellular vesicles in vitro
or as a calcium sequestrator on the shell-facing ectodermal cell surface in vivo (see
below for further discussion).
Our recent finding (Tuan & Knowles, 1984) of a plasma membrane Ca2+-activated
ATPase, which is also a near neighbour of the CaBP in the CAM, has raised the
possibility that the enzyme i9 somehow involved in the transport process, particularly
since it shares some properties with the many plasma membrane Ca2+-pumping
ATPases reported in various systems (Penniston, 1982; Schatzmann, 1982). The
findings reported here are clearly consistent with this notion, since enzyme inhibitors
also inhibit calcium uptake. However, since an 'ouabain-like', specific inhibitor for
plasma membrane Ca2+-ATPase has not been found, a specific one-to-one correspondence is not possible and the functional importance of the enzyme in calcium
uptake remains to be elucidated. It should be pointed out that calcium uptake
measured here in vitro exhibits Km values of 0-3-0-5 mM-Ca2+, which correspond
well with the lower affinity (Km 0-3 mM) of the enzyme (Tuan & Knowles, 1984).
For the purpose of exploring further experimental means of analysis, it is useful to
postulate, on the basis of currently available information and analogy with other
plasma membrane calcium pumps (Terepka et al. 1976; Penniston, 1982), how the
CAM calcium translocating mechanism may be assembled. As stated above, we postulate that subcellularly fractionated, calcium-uptake-competent microsomal vesicles
are inside-out structures formed from plasma membranes and contain at least the
CaBP and the Ca2+-ATPase, the former located in the internal space of the vesicles
whereas the latter is an integral membrane protein oriented so that ATP hydrolysis is
coupled to inward translocation of calcium. This inside-out orientation is therefore
consistent with the observed requirement of high external K + (i.e. cytosol-like
conditions) for functional uptake as reported in the accompanying paper. We believe
that this hypothetical model adequately accounts for the characteristics of the in vitro
microsomal calcium-uptake process. On a cellular level, Terepka et al. (1976) and we
Calcium transport by chorioallantoic membrane. II
95
Fig. 4. Ultrastructure of CAM ectoderm. Electron microscopy of perivascular cells of
the ectoderm revealed significant numbers of 'endocytotic-like' pits (white arrowheads)
and vesicles (black arrowheads) in various regions that directly line the shell membrane.
Also note the presence of abundant pinocytotic pits and vesicles in the expectedly
endocytotic endothelial cell, sm, shell membrane; ec, perivascular cell of the ectoderm;
en, endothelial cell. Bar, 100nm.
(Tuan et al. 19786) have previously postulated a pinocytosis model to explain
transcellular calcium translocation in the CAM. In line with this model, the CaBP
may be postulated as serving the role of a cell surface 'calcium-specific receptor',
which upon calcium binding triggers specific, adsorptive endocytosis leading to the
formation of pinocytic vesicles (Roth & Woods, 1982) that contain the CaBP and the
membranous Ca2+-ATPase, in a manner similar to that in the cell-free microsomal
vesicles studied here. The Ca2+-ATPase is orientated in situ so that it is capable
of continuously pumping Ca2+ inwards into the pinocytic vesicle and thereby
safeguarding its calcium load. Finally, by an unknown mechanism, perhaps by
acidification (Brown et al. 1983) or other ionic changes, the Ca 2+ -CaBP complex is
later dissociated, so that the calcium load may be delivered to the serosal side of the
perivascular processes of the ectodermal cells, whereas component(s) of the transport
apparatus, e.g. the CaBP, may be re-cycled to the proper region of the plasma
membrane facing the calcium source. It is at least partly consistent with this
hypothetical mechanism that the ultrastructure of the perivascular cells of the CAM
ectoderm reveals a significant number of 'pinocytotic-like' vesicles (Fig. 4), previously also observed by many other investigators (e.g. see Ganote et al. 1964;
Coleman & Terepka, 1972a; Dunn & Fitzharris, 1979). Our current efforts are
directed towards the testing of each of these steps to evaluate the validity of the
proposed mechanism.
This work was supported in part by grants from the National Institutes of Health (HD 15306,
HD 15822, and HD 17887) and the National Foundation/March of Dimes Birth Defects
Foundation (Basil O'Connor Starter Research grant 5-343 and Basic Research grant 1-939).
96
R. S. Tuan and others
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{Received 17July 1985 -Accepted2 October 1985)