(Received for publication, June 20, 1995; and in revised form, July 17, 1995)
From the
The synthesis of Calcium Green C, a lipophilic
fluorescent calcium-sensitive dye, and its use as a monitor of
Ca
efflux from cells is described. This indicator
consists of a Calcium Green-1 molecule conjugated to a lipophilic
18-carbon alkyl chain which will intercalate into cell membranes. The K
of the indicator for Ca
in aqueous solution (pH 7.2, 22 °C, ionic strength 0.1 M) is 0.23 ± 0.04 µM and in the presence
of liposomes is 0.062 ± 0.007 µM. Due to its high
negativity, the calcium chelating fluorophore faces the cell exterior,
when loaded under a defined set of conditions. The dye was found
largely on the surface of the cells when loaded at a concentration of 5
µM for 10 min at 37 °C. Five minutes after
introduction of EGTA, 83-95% fluorescence disappeared, indicating
that most of the fluorophore was on the cell surface. Photobleaching
was minimal (3-13%). A confocal laser scanning microscope was
used to detect and quantify fluorescence. Internalized dye was apparent
in cells loaded for longer times (30-60 min) and in
membrane-impaired cells, as shown by uptake of propidium iodide. Under
defined confocal laser scanning microscope settings, a transient
fluorescence at the periphery of
30% of the cells was observed
following 10
M parathyroid hormone
treatment, indicating the presence of outwardly directed calcium
transport across the plasma membrane. Calcium efflux usually lasted
7-10 min, peaking at around 2-3 min. Changes in cell shape
were also observed. Calcium efflux was shown to be sensitive to (a) 10 µM quercetin and 10 µM vanadate, partially specific inhibitors of plasma membrane
Ca
-ATPase, to (b) 0.1 mM trifluoperazine, an agent which renders calmodulin ineffective,
and to (c) 10 mM neomycin sulfate, which blocks
release of Ca
from intracellular stores. Thapsigargin
(5 µM), an inhibitor of Ca
-ATPase of the
endoplasmic reticulum, prolonged fluorescence. These observations
indicate that cell surface fluorescence was due to the capture of
Ca
by Calcium Green C
after
Ca
had been translocated across osteoblast plasma
membranes. Involvement of the plasma membrane
Ca
-ATPase, known to be present in osteoblasts in
substantial amounts, is implicated.
Osteoblasts line bone surfaces at sites of bone formation and
secrete a protein matrix that has the capacity to calcify. The precise
manner by which Ca passes through the layer of
osteoblasts to sites of calcification is not known. The demonstration
of substantial amounts of a plasma membrane Ca
-ATPase
in osteoblasts (Akisaka et al., 1988) raised the interesting
possibility that the enzyme may be involved in movement of
Ca
to calcifying sites in bone. More recently, a
plasma membrane Ca
-ATPase has been cloned and
sequenced from human osteoblast cell lines (SAOS-2; HOBIT) and shown to
bear high sequence homology to other plasma membrane
Ca
-ATPases (Kumar et al., 1993). In a
preparation of vesicles derived from osteoblast plasma membranes, the
magnitude and direction of calcium translocation has been determined
(Gay and Lloyd, 1995). Sealed inside-out vesicles took up
Ca
, implying that the direction of pumping in cells
was outward, as has been found for numerous other cell types (Carafoli,
1987; Garrahan and Rega, 1990). The uptake rate by osteoblast plasma
membrane vesicles was 9.9 ± 2.3 nmol/mg of protein/min, which
falls in the range reported for several other vesicle studies,
including red blood cells (Caride et al., 1983), parotid gland
(Takuma et al., 1985), neutrophils (Ochs and Reed, 1983), and
liver (Kraus-Friedmann et al., 1982).
Na
/Ca
exchange also exists in
osteoblasts (Krieger, 1992; Short et al., 1994) and may
contribute in a minor way to the Ca
efflux observed,
as subsequently discussed.
The present study was undertaken to
establish characteristics of the fluorophore, Calcium Green
C, when applied to living cells and to utilize the dye to
evaluate calcium efflux in intact osteoblasts. Parathyroid hormone was
employed to stimulate an increase in
. (
)This occurs as a consequence of Ca
release from intracellular stores as has been shown in other
studies of osteoblasts (e.g. Reid et al.(1987),
Civitelli et al.(1989)). It was anticipated that the return of
[Ca
]
to prestimulatory
levels would involve, in part, efflux by plasma membrane
Ca
-ATPase activity, since this enzyme system is a
major pathway utilized by cells to restore
[Ca
]
to basal levels
(Carafoli et al., 1990). The lipophilic calcium-sensitive
fluorescent dye, Calcium Green C
, was selected as a likely
candidate for monitoring Ca
efflux. Since the
fluorophore is hydrophilic and is negatively charged, it is excluded
from the lipid bilayer; the lipophilic hydrocarbon chain lodges in the
plasma membrane, holding the dye in close proximity to the membrane. We
reasoned that Ca
, upon emerging from the cell, would
be trapped by the fluorophore, which would then fluoresce. A laser
scanning confocal imaging system was used to visualize sites of calcium
efflux and to measure both the intensity and duration of Ca
efflux. Comparisons with another lipophilic calcium-sensitive
dye, C
-Fura-2, previously reported by Etter et
al.(1994) are included under ``Discussion.''
Based on previous
experience with the synthesis of a lipophilic Fura-2 (Etter et
al., 1994), we synthesized a lipophilic derivative of Calcium
Green-1. The synthesis starts with catalytic reduction of
5,5-dinitro-BAPTA tetramethyl ester to the diamine (Pethig et
al., 1989), which is then reacted with 1 eq of stearoyl chloride
to give the monosubstituted lipophilic BAPTA tetramethyl ester. This is
conjugated to a protected dichlorofluorescein, and the protecting
groups on the BAPTA and dichlorofluorescein are hydrolyzed with base.
The resulting soapy fluorescent product is purified by reverse-phase
chromatography to give pure Calcium Green C tetrapotassium
salt. The synthesis of Calcium Green C
tetrapotassium salt
requires synthesis of four intermediates, shown in Fig. 1.
Figure 1:
Synthetic pathway leading to Calcium
Green C. Roman numerals correspond to the
compounds whose synthesis is detailed under
``Experimental Procedures.''
Introduction of Calcium Green C into the plasma
membranes involved first rinsing osteoblasts adherent to coverslips
three times in a reduced calcium balanced salt solution (RCBSS) which
contained 127 mM NaCl, 3.8 mM KCl, 1.2 mM KH
PO
, 0.8 mM MgCl
, 5
mM glucose, and 10 mM HEPES buffer at pH 7.3. The
cells were then exposed to 1, 2, 5, or 10 µM Calcium Green
C
, initially dissolved in anhydrous dimethyl sulfoxide,
then diluted in RCBSS for 1, 2, 5, 10, 30, or 60 min at room
temperature. Cells were rinsed again three times with RCBSS to remove
excess indicator. The coverslip was inverted and placed in a
Dvorak-Stotler Controlled-Environment Culture Chamber (DSC200,
Nicholson Precision Instrument, Gaithersburg, MD) that was preloaded
with RCBSS. The 5 µM concentration (with a final
concentration of dimethyl sulfoxide at 0.5%) and 10-min loading time
were found to be optimal. Propidium iodide (2 µg/ml) was present in
the RCBSS in some experiments as a test of cell viability.
In order to assess the extent of photobleaching, cells were
loaded with 5 µM Calcium Green C for 10 min,
then placed in buffer that contained 1.2 mM CaCl
,
and exposed to the laser beam every 5 s for 5 min (Table 1).
Quercetin, sodium orthovanadate, neomycin sulfate, trifluoperazine, thapsigargin, and HEPES buffer were obtained from Sigma. PTH (bovine, 1-84) was obtained from the National Hormone and Pituitary Agency (Bethesda, MD).
Figure 2:
Response of Calcium Green C to Ca
in aqueous solution. This figure shows
the emission response of Calcium Green C
(1
µM) in the presence of increasing
[Ca
]. The buffer solutions have free
Ca
concentrations from ``zero'' (10 mM EGTA) to 1.35 µM (9 mM CaEGTA, 1 mM
K
EGTA). The ionic strength of the calibration buffers is
0.1 M (100 mM KCl), and they are buffered to pH 7.20
with 10 mM MOPS. The excitation is at 488 nm with the emission
scanned from 490 nm to 575 nm.
Figure 3:
Fig. 3. A, osteoblasts were
loaded with Calcium Green C in RCBSS followed by addition
of 1.2 mM CaCl
to the solution bathing the cells. B, the same cell treated with 50 mM EGTA for 5 min.
The fluorescence at the cell periphery (arrows) has largely
disappeared. Scale bar = 25 µm.
Fig. 4. These
are three optical sections from a series of sections in the x-yplane, parallel to the coverslip of a cell loaded
with Calcium Green Cand exposed to 1.2
mMCaCl
. The rings of
fluorescence diminished in size as the optical sections progressed
through the cell. A, section close to the coverslip; B, section through the cell center; C, section at the
tip of the cell distal to the coverslip. The decreasing diameter of the
cell is evident. Scale bar = 25 µm.
Fig. 5. A
series of fluorescence changes as a function of time in Calcium Green
C-loaded osteoblasts following
10
MPTH application. The
increase in fluorescence usually began
10 s after PTH treatment,
peaked around 3 min, and gradually decreased in about 7-10 min.
Note changes in cell shape, for example at arrows. Time
following PTH application was 5 s (A), 10 s (B), 50 s (C), 150 s (D), 250 s (E), and 540 s (F). Scale bar = 50
µm.
Quercetin, sodium vanadate, trifluoperazine, and neomycin
sulfate substantially impaired Ca efflux, both in
magnitude and duration (Table 3). Thapsigargin caused the
fluorescence to to persist for 15-17 min (Table 3), nearly
twice the time found for untreated controls.
In this paper we have described the synthesis and spectral
properties of a lipophilic long-chain Ca-sensitive
fluorescent dye, Calcium Green C
, that localizes
preferentially in the plasma membrane of cells. Our goal was to utilize
this fluorescent Ca
-specific probe to monitor
Ca
translocation out of osteoblasts. The
Ca
-trapping head group of Calcium Green C
facing the external medium served as a reporter of Ca
efflux. Conjugation of Calcium Green-1 with a lipophilic tail, 18
carbons long, as shown in Fig. 1, provided a means of situating
the fluorescent probe along a lipid-based membrane while preserving the
capacity of the indicator to fluoresce. It is likely that the method of
loading the dye achieved placement of most of the dye into the plasma
membrane with the fluorophore facing the outward direction since added
external EGTA abolished fluorescence. Furthermore, the observation that
the plasma membrane Ca
-ATPase inhibitors, quercetin,
vanadate and trifluoperazine, greatly reduced membrane fluorescence (Table 3) indicates that most of the Calcium Green C
was loaded in the preferred orientation. If substantial amounts
of Calcium Green C
had been inwardly oriented, the dye
would have fluoresced when [Ca
]
increased whether Ca
efflux was inhibited or
not. While some fluorescence was observed in the cytosol, it was minor
compared to the cell periphery under short (10 min) loading times. At
the longer loading times, internal fluorescence was common. Using
three-dimensional imaging techniques Calcium Green C
has
been found useful for demonstrating Ca
efflux from
primary osteoblasts stimulated with parathyroid hormone. It was also
possible to monitor the duration of the response. This is the first
study to demonstrate that Calcium Green C
can be used to
detect Ca
efflux from cells.
The spectral response
of Calcium Green C to increasing concentrations of
Ca
in aqueous solution, where the K
was 0.23 µM, shows a linear increase in emission
intensity in the submicromolar range (Fig. 2). In this state,
the lipid tail of the dye is not embedded in a membrane, so the
response is expected to differ slightly from that in cells. In the
presence of DOPC liposomes (spectra not shown), a linear increase in
fluorescence with increasing concentrations of Ca
was
also observed, but the affinity of the dye for Ca
was
higher (K
= 0.062 µM). The
water solubility of this probe is better than for
C
-Fura-2, as Calcium Green-1 has an extra negative charge.
This should facilitate loading the dye into cell membranes from an
aqueous solution. Further, once in place in the membrane, dye-dye
interactions would be reduced due to the negativity of the head group.
In cultured osteoblasts, Calcium Green C appears to be
localized along the entire plasma membrane, except for regions
associated with the coverslip. Ca
specificity is
indicated since the calcium greens have been shown to preferentially
bind calcium (Girard et al., 1992). Further, little
fluorescence was observed under the chosen confocal settings when cells
were bathed with RCBSS, which intrinsically contained trace amounts of
Ca
. Fluorescence was induced when Ca
was added to the RCBSS (Fig. 3A) and then largely
disappeared when the Ca
-specific chelating agent EGTA
was added externally (Fig. 3B). Because of the special
orientation of Calcium Green C
and its relatively low K
, it is critical that trace amounts of calcium
present in the RCBSS be small. At a Ca
concentration
present in the RCBSS of 0.22 µM,
50% of the indicator
would be free to bind calcium emerging from the cell. The K
measured in aqueous solution, rather than in
liposomes, is considered to better represent the K
of the dye intercalated into the plasma membrane since the
fluorescent head group is entirely extracellular and is bathed in
aqueous solution.
Another near-membrane Ca concentration indicator has been described recently that consists
of Fura-2 conjugated to a lipophilic C
alkyl chain (Etter et al., 1994). C
-Fura-2 orients in the plasma
membrane so that the fluorophore faces from the side to which it was
applied. This indicator was found to detect rapid, localized changes in
[Ca
]
which are undetectable by
water-soluble bulk cytosolic fluorescent Ca
indicators. Calcium Green C
, on the other hand, has
a higher negative charge, which would assist in maintaining the outward
orientation of the dye in the plasma membrane due to the negative
membrane potential of intact cells. In addition, the negativity of
Calcium Green C
would result in fewer dye-dye interactions
once the dye is positioned in the cell membrane. The calcium greens are
useful for kinetic analysis due to increased quantum yields on binding
Ca
(Haugland, 1993). However, since the calcium
greens are not ratiometric dyes, they are not useful for measuring ion
concentrations accurately. The lower K
of Calcium
Green C
in liposomes than for C
-Fura-2 (0.062
µMversus 0.15 µM) indicates that
Calcium Green C
can be used to detect lower levels of
Ca
and therefore is more sensitive. In this study we
did not use an EGTA/CaEGTA buffer in the assay medium surrounding the
cells because of concerns with cell membrane instability. Use of such a
buffering system, needs to be explored, however, for detection of very
low levels of Ca
. The improved sensitivity of the
calcium greens has been discussed by Eberhard and Erne(1991). In
addition, the calcium greens show increased specificity, speed of
response, and higher spatial and temporal resolution (Haugland, 1992).
Advantages of Calcium Green C
being excitable at longer
wavelengths over that of C
-Fura-2 include less cellular
photodamage and decreased autofluorescence. In addition, Fura-2 may
bind to cellular proteins and could lead to alterations in the response
of the indicator to Ca
(Blatter and Wier, 1990).
The present study is the first direct demonstration of
Ca efflux from osteoblasts. The source of
Ca
is believed to be from intracellular stores since
cytosolic calcium in osteoblast cell lines has been shown to increase
following PTH treatment (Reid et al., 1987; van Leeuwen et
al., 1988; Bidwell et al., 1991; Yamaguchi et
al., 1991). The pathways by which increased
[Ca
]
is restored to basal
levels are incompletely understood but are believed to involve
generation of IP
(Ferris and Snyder, 1992). In the present
study, we have shown that a portion of increased
[Ca
]
is handled by a plasma
membrane Ca
-ATPase-dependent efflux mechanism and
that re-entry into intracellular stores formed by the endoplasmic
reticulum also occurs. By blocking the latter process with thapsigargin
(Thastrup et al., 1987), more cytosolic calcium was available
for translocation to the cell exterior as shown by a prolonged
fluorescence at the cell surface. An alternative explanation for the
prolonged effect could be that ATP-dependent processes in the plasma
membrane were partially inhibited by thapsigargin. This possibility is
contraindicated by studies which show the specificity of thapsigargin
for Ca
-ATPase of the endoplasmic reticulum (Lytton et al., 1991). It has been shown that once PTH binds to
receptors on the cell membrane, phospholipase C is activated (Lowik et al., 1985). This in turn generates IP
which
will then bind to the endoplasmic reticulum membrane derived
Ca
storage compartment, causing Ca
to be released. Our experiments show that a substantially reduced
amount of Ca
efflux occurred in the presence of
neomycin sulfate, an inhibitor of phospholipase C and, therefore,
IP
generation (Bidwell et al., 1991). This
supports the view that the Ca
that is secreted in
response to PTH is derived mainly from intracellular stores, through
the IP
pathway.
Two major Ca efflux
systems are known to exist in the plasma membrane of cells, the
ubiquitous Ca
-ATPase (Carafoli et al.,
1990), and the less widespread Na
/Ca
exchanger (Carafoli, 1987). The plasma membrane
Ca
-ATPase has been shown to be present in osteoblasts
in substantial amounts (Akisaka et al., 1988; Kumar et
al., 1993; Gay and Lloyd, 1995).
Na
/Ca
exchange has also been
demonstrated in osteoblasts, but at an unknown level of participation
(Krieger, 1992; Short et al., 1994). Typically, excitable
tissues have notably high levels of the exchanger, whereas nonexcitable
tissues have less or even no Na
/Ca
exchange (Philipson and Nicoll, 1993). The extent to which the
Ca
-ATPase and Na
/Ca
exchange systems may have contributed to this study can be
partially assessed from the data in Table 3. The three inhibitors
used, trifluoperazine, vanadate, and quercetin, all markedly affect the
Ca
-ATPase.
Na
/K
-ATPase, the enzyme which drives
Na
/Ca
exchange by creating a sodium
gradient, is also affected by these inhibitors. However, while
trifluoperazine has been shown to inhibit
Na
/K
-ATPase activity in red blood
cell hemolysates, at the concentration we used (0.1 mM),
trifluoperazine is more effective (2-5
) in reducing
Ca
-ATPase activity (Luthra, 1982). In ameloblasts,
cells which also form a calcifiable matrix, trifluoperazine has no
effect on Na
/K
-ATPase activity
(Sasaki and Garant, 1987). Quercetin, at the concentration we used (10
µM), has almost no effect on eel
Na
/K
-ATPase (Kuriki and Racker,
1976), but has a K
= 4-6 µM for Ca
-ATPase of erythocytes
(Wüthrich and Schatzmann, 1980). Vanadate is also
notably effective for red blood cell Ca
-ATPase having
a K
= 5 µM (Niggli et
al., 1981). Under some conditions, however, vanadate has been
shown to more effective for
Na
/K
-ATPase than for
Ca
-ATPase (Bond and Hudgins, 1980). Our finding of
similar degrees of inhibition of Ca
efflux with all
three inhibitors, which have widely differing degrees of effectiveness
on Na
/K
-ATPase, suggests that
Na
/Ca
exchange may have made little
to no contribution to the Ca
-efflux monitored in this
study. Further, PTH may have inhibited
Na
/K
-ATPase activity as has been
shown in the renal tubule (Ribeiro and Mandel, 1992). However,
manipulation of external Na
and treatment with
ouabain, a specific Na
/K
-ATPase
inhibitor, are needed for critical assessment of the participation of
Na
/Ca
exchange.
It has been reported that parathyroid hormone alters the shape of osteoblasts, usually from a spreadout to a stellate morphology (Egan et al., 1991; Ali et al., 1990). Our observations of cell shape change induced by PTH corroborate the earlier reports.
In
summary, this study has provided evidence that Calcium Green C can be employed to monitor Ca
efflux by cells.
Plasma membrane Ca
-ATPase of primary osteoblasts is
implicated as having a major role in actively transporting
intracellular calcium to the extracellular space. The enzyme activity
is calmodulin-dependent and can be partially blocked by
Ca
-ATPase inhibitors. Also shown is that calcium is
released from intracellular stores which utilize the IP
pathway upon receiving signals from PTH-occupied receptors; this
process is substantially impaired by interfering with IP
production. Apparently, when thapsigargin blocks re-entry of
intracellular calcium into the endoplasmic reticulum, more calcium is
transported through the plasma membrane to the exterior.