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(Received for publication, June 20, 1995; and in revised form, July 17, 1995) From the
The synthesis of Calcium Green C
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 The present study was undertaken to
establish characteristics of the fluorophore, Calcium Green
C
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
Figure 1:
Synthetic pathway leading to Calcium
Green C
Introduction of Calcium Green C
In order to assess the extent of photobleaching, cells were
loaded with 5 µM Calcium Green C
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
Figure 3:
Fig. 3. A, osteoblasts were
loaded with Calcium Green C 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 C Fig. 5. A
series of fluorescence changes as a function of time in Calcium Green
C
Quercetin, sodium vanadate, trifluoperazine, and neomycin
sulfate substantially impaired Ca
In this paper we have described the synthesis and spectral
properties of a lipophilic long-chain Ca The spectral response
of Calcium Green C In cultured osteoblasts, Calcium Green C Another near-membrane Ca The present study is the first direct demonstration of
Ca Two major Ca 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
Volume 270,
Number 38,
Issue of September 22, pp. 22445-22451, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 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.
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.
, 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.''
Synthesis of Calcium Green C
Calcium Green-1 is a fluorescent
indicator of free Ca,
Tetrapotassium Salt ions. It consists of a
dichlorofluorescein conjugated to an aromatic Ca
binding site (BAPTA) through an amide spacer (Haugland, 1992).
This indicator can be excited at 488 nm and gives an increase in
emission intensity at 530 nm on reversibly binding
Ca
, with a K
for Ca of 0.189 µM. The increase in emission intensity for
free Calcium Green-1 in solution is approximately 12-fold.
Dichlorofluorescein was chosen as the fluorophore for the synthesis of
Calcium Green because it is less pH-sensitive than fluorescein itself
and still retains a high quantum yield (0.75 at saturating
Ca
) when excited at 488 nm.
tetrapotassium
salt. The synthesis of Calcium Green C tetrapotassium salt
requires synthesis of four intermediates, shown in Fig. 1.
. Roman numerals correspond to the
compounds whose synthesis is detailed under
``Experimental Procedures.''
Synthesis of Compound I: 5,5`-bis-Amino-BAPTA Tetramethyl
Ester
A solution of 11 g (17.7 mmol) of 5,5`-dinitro-BAPTA
tetramethyl ester as described by Pethig et al.(1989) is
dissolved in 200 ml of DMF and shaken under hydrogen at 40 p.s.i. for 3
h in the presence of 0.8 g of 10% palladium on charcoal. TLC in ethyl
acetate shows the formation of a single new product that is colorless
and gives a brown/red product on reaction with ninhydrin. The catalyst
is removed by filtration through diatomaceous earth, and the colorless
filtrate is concentrated under vacuum to a gray oil, which is stirred
with 200 ml of methanol for 1 h to give a gray solid. The solid is
filtered and dried to give 7.0 g (88% yield), pure to TLC in ethyl
acetate or 5% methanol in chloroform.Synthesis of Compound II: N-Stearoyl-5,5`-bis-amino-BAPTA
Tetramethyl Ester
A solution of stearoyl chloride (1.6 g; 5.4
mmol) in 20 ml of dry DMF is added dropwise over 2 h to a stirring
solution of compound I (3.0 g, 5.4 mmol) in 70 ml of dry DMF. TLC
analysis in ethyl acetate shows the formation of two UV quenching
products. The predominant product runs with an R
of 0.4 and reacts with ninhydrin to give a ruddy brown product.
The reaction is added to 150 ml of ethyl acetate and washed three times
with saturated NaCl and once with pure water. The organic layer is
dried over Na
SO
and evaporated to a tan oil.
This is purified over 150 ml of silica gel (40-60 µm) eluted
with 20% ethyl acetate, 80% chloroform. Purified fractions are pooled
and evaporated to a semicrystalline solid that is recrystallized from
methanol to give 1.4 g of pure material (40% yield). NMR in CDCl
shows 0.95 ppm of H, t; 1.4 ppm of 27H, s(b); 1.7 ppm
of 2H, m; 2.4 ppm of 2H, m; 3.6 ppm of 12H, s; 4.1 ppm of 4H, s; 4.15
ppm of 4H, s; 4.2-4.4 ppm of 4H, d; 6.8 ppm of 2H, d; 7.05 ppm of
2H, d; 7.3 ppm of 2H, s.Synthesis of Compound III:
5-Carboxy-2,7-dichlorofluorescein Diacetate, Isobutyl
Anhydride
The 5-isomer of carboxy-2,7-dichlorofluorescein
diacetate is purified from mixed 5 and 6 isomers (Molecular Probes) by
repeated crystallization from ethanol. The resulting colorless solid is
dissolved in 20 ml of CHCl at 0 °C with
isobutyl chloroformate (4.67 mmol) added. The reaction is stirred for 1
h and is evaporated to a colorless semisolid; ethyl acetate (50 ml) is
added, and the insoluble salts are removed by filtration. The filtrate
is evaporated and dried for 5 h under high vacuum to give the isobutyl
anhydride as a colorless foam (2.2 g; 3.5 mmol, 90% yield). NMR in
CDCl shows 1.55 ppm of 6H, s; 2.35 ppm of 6H, s; 3.95 ppm
of 1H, s; 4.4 ppm of 2H, q; 6.85 ppm of 2H, s; 7.2 ppm of 2H, s; 7.85
ppm of 1H, s; 8.15 ppm of 1H, d; 8.35 ppm, m.Synthesis of Compound IV: Calcium Green C
A solution of Compound III
(0.23 g; 0.36 mmol) in 1.5 ml of dry DMF is added dropwise over 0.5 h
to a stirring solution of Compound II (0.3 g, 0.36 mmol) in 1.5 ml of
dry DMF at 0 °C. After stirring at this temperature for 2 h, TLC in
5% methanol, 95% chloroform shows conversion of the two starting
materials to a new product of intermediate RDiacetate, Tetramethyl Ester. On
exposure to ammonia fumes, the product turns dark red and is only
weakly fluorescent. The reaction is evaporated at 15 °C to a tan
oil, which is purified on silica gel (40-60 µm) eluted with
pure ethyl acetate. The pure fractions are combined and evaporated to a
tan oil with a mass of 250 mg.Synthesis of Compound V: Calcium Green
C
Compound IV (0.25 g,
0.19 mmol) is dissolved in 1 ml of dioxane and diluted with 2 ml of
methanol. Potassium hydroxide (0.11 g, 1.9 mmol) is dissolved in 0.5 ml
of pure water and added to the stirring ester over 10 min. The solution
immediately turns red as the acetates are cleaved from the
dichlorofluorescein. The reaction is stirred at room temperature for 20
h until TLC in 1:1 CHCl, Hexapotassium Salt:CHOH shows a single
fluorescent product with an R of 0.5. Cleavage
products are also visible as minor impurities formed in the hydrolysis.
The reaction is evaporated, diluted with 5 ml of pure water, and
purified twice on 300 ml wet bed volume of lipophilic Sephadex LH-20
eluted with pure water adjusted to pH 8 with the addition of dilute
KOH. Pure fractions are combined, frozen, and lyophilized to give 210
mg of red powder. NMR in DO shows 0.7 ppm of 3H, t;
1.0-1.5 ppm of 28H, s(b); 1.6 ppm of 2H, m(b); 2.4 ppm of 2H,
s(b); 3.9 ppm of 4H, s; 4.0 ppm of 4H, s; 4.5 ppm of 4H, s; 6.75 ppm of
3H, s; 6.85 ppm of 1H, d; 6.9 ppm of 1H, d; 7.05 ppm of 1H, d; 7.15 ppm
of 1H, s; 7.3-7.4 ppm of 2H, m; 7.4 ppm of 2H, s; 7.45 ppm of 1H,
d; 8.15 ppm of 1H, d; 8.45 ppm of 1H, s.Spectral Properties of Calcium Green
C
Dissociation constants for Ca were determined in EGTA/CaEGTA-buffered Ca
solutions with free Ca
concentrations varying
between 0 and 1.35 µM in 100 mM KCl and 10 mM MOPS (pH 7.2, 22 °C) using the Calcium Calibration Kit I
(Molecular Probes) and an MPF-44B fluorescence spectrophotometer
(Perkin-Elmer); excitation, 506 nm; emission, 526 nm; slit widths, 5
nm. Calcium buffers were made by the ``pHimetric'' method of
Tsien and Pozzan(1989). Calcium Green C
was tested for
response to free Ca both in aqueous solution and in
the presence of DOPC liposomes at a dye concentration of 1
µM. To create liposomes, 100 µl of 40 mM DOPC
(in ethanol) was injected slowly (over 1 min) into vortexing buffers,
one that contained 10 mM CaEGTA and one that contained 10
mM EGTA, using a Hamilton Syringe and 23-gauge needle. The
buffered DOPC solutions were stirred slowly for 15 min. Calcium Green
C
in anhydrous dimethyl sulfoxide (1 mM) was then
added to both DOPC solutions to achieve a dye to DOPC ratio of 1:400.
These solutions were stirred for 45 min to ensure an even distribution
of the dye and DOPC, then mixed in graded proportions according to
Tsien and Pozzan(1989). The response and affinity of the indicator is
dependent on the environment, with an increase in emission intensity of
9-fold in aqueous solution and 3.5-fold in the presence of liposomes. A
plot of log(F - F
/F
- F) versus log [Ca] gives the
K
) of the indicator (pH 7.2, 22 °C,
ionic strength 0.1 M) as 0.23 ± 0.04 µM in
aqueous solution and 0.062 ± 0.007 µM in the
presence of DOPC liposomes.
Defining Conditions for Loading Calcium Green C
Primary osteoblasts were
isolated as described previously (Gay et al., 1994). Briefly,
cells were scraped from periosteal surfaces of 2-3-week-old chick
tibias, after mild enzyme treatment, and cultured on 25-mm glass
coverslips in Dulbecco's modified Eagle's medium (Sigma)
plus 10% heat-inactivated fetal bovine serum for 6 to 8 days. into Osteoblasts 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 KHPO, 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.Determination of [Ca
Fluo-3 (1
mM, Molecular Probes) was dissolved in anhydrous dimethyl
sulfoxide and further diluted to 0.1 mM with RCBSS or with
distilled, deionized H] in
the Reduced Calcium Balanced Salt Solution
O. Fluo-3 (10 µl, 0.1
mM) was added to 500 µl of RCBSS in a 0.7-ml microcell
cuvette (type 4, NSG Precision Cell, Inc., Farmingdale, NY), and
fluorescence was measured (MPF-44B, Perkin-Elmer; excitation, 506 nm;
emission, 526 nm; slit widths, 5 nm). The calcium concentration was
determined using a standard Ca concentration curve (0
to 39.8 µM Ca
) generated from the
Calcium Calibration Buffer Kit II (Molecular Probes). This analysis
revealed that the concentration of Ca
in RCBSS was
0.22 µM.
Determining Specificity of Membrane Localization and
Extent of Photobleaching
In order to demonstrate that the dye
was localized specifically in the plasma membrane, Calcium Green
C-loaded cells were exposed to 1.2 mM CaC1 added to RCBSS which was added through a port in the controlled
environment chamber. Patterns of fluorescence were observed in optical
sections using the 488 nm laser line, zoom 1.0, 10% neutral density
filter, aperture opening 2.7 mm, gain 8, and black level 2 settings on
the confocal microscope (Bio-Rad MRC 600). For a control, cells were
treated with 50 mM EGTA for 5 min in order to chelate added
calcium. 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).
Characterization of Ca
In order to characterize CaEfflux in
Osteoblasts Using Calcium Green C
As an
Ion Trap
efflux by osteoblasts, cells loaded with Calcium Green C
were placed in the Dvorak-Stotler Chamber in RCBSS, and a bolus
(20 µl) of PTH (final concentration 10M) was injected through a port into the chamber as a
means of increasing [Ca
]
.
Optical sections, 1.0 micron in thickness, in the x-y plane through cell centers were recorded every 5 s for the first
200 s, then every 60 s thereafter, depending on the duration of the
fluorescence due to Ca efflux. Under the chosen
settings for the confocal system, the background fluorescence caused by
the trace amount of Ca
present in RCBSS was
negligible. Controls included: thapsigargin (5 µM), an
inhibitor of endoplasmic reticulum Ca
-ATPase;
trifluoperazine (0.1 mM), an inactivator of calmodulin;
quercetin (10 µM) and sodium vanadate (10 mM),
inhibitors of plasma membrane Ca
-ATPase; and neomycin
sulfate (10 mM), an inhibitor of phospholipase C and therefore
of generation of IP
, the intracellular signal that causes
Ca release from intracellular stores. Cells were
pre-incubated with the inhibitors in RCBSS for 10 min at room
temperature. RCBSS in the culture chamber also contained the same
concentration of each inhibitor. Fluorescence images were recorded, and
image analysis was carried out using COMOS commands in the Bio-Rad
software package. The image space was divided into pixels (0.17
µm
when using the 40
objective lens) and
assigned a gray level (on a scale of 1-256) as a measure of
fluorescence intensity.
Synthesis and Fluorescence Excitation Spectra of
Calcium Green C
The structures of the compounds
synthesized, then utilized in the synthesis of Calcium Green
C, described under ``Experimental Procedures,''
are diagramed in Fig. 1. Fig. 2shows the excitation
spectra of 1 µM Calcium Green C in the
presence of increasing concentrations of Ca in
aqueous solution. As [Ca
] increased, the
emission intensity (at 530 nm) increased with little shift in
wavelength.
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.
Localization and Characterization of the Lipophilic
Calcium Green C
Cells loaded with
Calcium Green Cin Cells that were exposed to 1.2 mM
CaCl appeared as rings in optical sections produced by
confocal microscopy (Fig. 3A). A low level of
cytoplasmic staining was also evident in many cells. This was, however,
a minor contribution to total cell fluorescence. Occasionally, cell
cytoplasm was intensely fluorescent; this phenomenon coincided with
cell death, as these cells were found to stain with propidium iodide.
Following the addition of EGTA to the observation chamber, fluorescence
of cell peripheries were markedly reduced (Fig. 3B; Table 2). Controls indicated that photobleaching contributed only
to a minor extent to the disappearance of the fluorescent images; as
shown in Table 1, loss of fluorescence due to photobleaching
after a total of 60 scans over a time frame of 5 min ranged between 3
and 13%. Serial optical sections along the z axis of
dye-loaded cells further revealed that Calcium Green C was
evenly located along peripheries of cells (Fig. 4).
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.and 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.
-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.
Characterization of Calcium Efflux
The plasma
membrane of approximately 30% of the 8-day-old cultured osteoblasts
were found to fluoresce following PTH stimulation. A rapid and
transient increase in fluorescence was observed on the surface of the
responding osteoblasts when PTH was added to the cell chamber (Fig. 5).
The initial efflux was usually detectable 10 s after the addition of
PTH. Fluorescence intensity peaked around 2-3 min and persisted
for about 7-10 min. Cell shape changes, including the appearance
of cell processes, were observed in some cases after PTH stimulation
(Fig. 5). 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.
-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.
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. 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. 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).
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. 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.
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.
)], cytosolic free Ca
concentration; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid; DMF, N,N-dimethylformamide; DOPC, L-
-dioleoylphosphatidylcholine; MOPS,
3-(N-morpholino)propanesulfonic acid; PTH, parathyroid hormone
(bovine, 1-84); RCBSS, reduced-calcium balanced salt solution;
IP, inositol 1,4,5-trisphosphate.
Virginia R. Gilman provided technical expertise and
Joseph P. Stains editorial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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