Vol.
183,
March
No.
2, 1992
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Paqes
16, 1992
IS THE
OSTEOCLAST
CALCIUM
C.M.R.
Bax, V. SShankar,
Department
*The
Received
of Cellular
Physiological
January
Elevated calcium
B. S. Moonga,
C. L.-H.
Huang*,
Sciences, St. George’s SW17 ORB , U .K.
University
CALCIUM
of Cambridge,
and
CHANNEL
?
M. Zaidi
Hospital
Medical
Cambridge
School,
CB2 3EG,
U . K.
1992
calcium levels presumably
whether or not Ca2+-induced a divalent cation channel. a distinct
A RECEPTOR-OPERATED
and Molecular London
Laboratory,
20,
extracellular levels ([Ca2+]i),
comprises
“RECEPTOR’
619-625
([Ca2+],) inhibit via the activation
cytosolic
free
It is unclear involves the direct gating, by the putative “receptor”, of that ICa2+]i elevation in response to elevated [Ca2+],
[Ca2+]i elevation The results show
component
osteoclast function by elevating of a surface Ca2+ “receptor”.
of Ca 2+ influx,
the magnitude
of which
can be decreased
and
increased,
respectively, by depolarising (100 mM-[K+]) and hyperpolarising (1 PM-[valinomycin]) the osteoclast membrane. In addition, activation of the putative Ca2+ “receptor” by elevated [Ca2+]ecauses influx of the related divalent cation, magnesium (Mg2+). We suggest that Ca2+ influx induced by Ca2+ “receptor” activation is a major component of the observed [Ca2+]i response. ~ 194.2 AC.3&rnlC Pm2SL. IIIC.
During high
the process
concentrations
the cell (1). directly
We have
regulate Ca2+
accompanied
with (3,4),
into
osteoclast
Ca2+
chief
cells
“receptor” other
divalent
receptors would
lowered
of depolarisation examined Mg2+-sensitive
the ambient (2).
release Ca2+
a rise of [CaZ+]i
cations.
upon and
the effects
that
present would
shown
is determined its membrane
cell retraction
bone
resorption
detects
a change
that
to that found explored
potential
of Ca2+
the influx
(review, upon
extracellular
magfura.
[Ca2+],induccd
(7).
of Ca2+,
induced
gradient
across therefore
[Ca2+]i ([Mg2+]3
In these
clear that
respects,
in response
attcmptcd
Ca2+
by Caz+
to stimulation
the cell membrane
the
secreting
putative
to its activation
elevation
it then
hormone the
3)
cell-matrix
(4, 6) which
or not
elevation.
(2,
becoming
on parathyroid
in response
9). We have
magnesium
(4), reduced
in [Ca2+],
site can
([Ca2+]i)
It is now
whether
of
to an elevated
Ca2+
mechanism
have
the influx
(2).
to
border
resorptive
osteoclasts
(2), including
by the electrochemical
hyperpolarisation of [Ca2+]eand
allow
rat
exposed
the ruffled
at the osteoclast
of isolated
is similar we
beneath
of cytosolic
or “receptor”
study,
and is consequently
an elevation
(5) and reduced
system
It has been
fluorochrome,
concentration
exposure
inhibition
generates
rise up to 30 mM
by a dihydropyridinc-insensitive
In the
a channel
Ca2+ The
sensor
detection-transduction 8).
may
causes
of functional
enzyme
the osteoclast these
([Ca*+]3
a unique
by agonists, depend
that
resorption, (Ca2+);
function
evidence
(review: gates
shown
concentration
in the rat osteoclast, transduces
calcium
osteoclast
extracellular
adhesion
of bone
of ionised
or of
and hence,
to study
the effects
In addition,
we have
on Mg2+
influx
using
the
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Osteoclast isolation and culture: Femora and tibiae obtained from neonatal rats were cleaned and cut across their epiphyses in N-2-hydroxyethyl piperazine-N’-2-ethane sulphonic acid (HEPESl-buffered Medium 199 supplemented with foetal calf serum (FCS) (10% v/v) tall from ICN Flow, UK). Osteoclasts were disaggregated by curetting bones into medium and agitating the suspension with a pipette. The supematant was then dispersed onto 22 mm glass coverslips (0 grade) and cells were allowed to sediment and attach for 30 minutes at 3X. Measurement
of [Ca2+]ii Osteoclasts, settled onto glass coverslips, were incubated with the ester of fura 2 (fura 2/AMl (10 PM) (Novobiochem, U.K.1 in Medium 199 for 40 minutes. The coverslips with the dye-loaded cells were then transferred to a perspex bath mounted onto the stage of a microspectrofluorimeter constructed from an inverted microscope (Diaphot; Nikon, Telford, U.K.). Osteoclasts, constantly bathed in buffered salt solution (BSS) containing 130 mM-[NaCl], 5 mM-[KCl], 1.25 mM-[CaCl2], 0.8 mM-[MgCl2], 5 mM-[glucose] and 10 mM-[HEPES] (pH - 7.4), were exposed alternately to excitation wavelengths of 340 and 380 nm using a computer-controlled spinning filter wheel. The fluorescent signal was directed to the sideport of the microscope which contained a 510 nm interference filter. The emission, obtained from single osteoclasts at the two excitation wavelengths, was fed into a photomultiplier tube (PM28B, Thorn EMI, Middlesex, U.K.). Single photon currents were then counted by a photon counter (Newcastle Photometric Systems, U.K.). Photons s-1 in each channel were recorded in an IBM microcomputer and the ratio of excitation intensities fF340/F380) calculated and displayed, [Ca2+]i was determined from a standard curve obtained by intracellular calibration (10). acetomethoxy
Measurement of [Mg2+]i : Osteoclasts, settled onto glass coverslips, were incubated with the acetomethoxy ester of magfura (magfura/AM) (1 l.rMl (Novobiochem, U.K.1 in Medium 199 for 30 minutes. The coverslips with the dye-loaded cells were then transferred onto the stage of a microspectrofluorimeter and incubated as described above. The emission intensity, following excitation of the cells at 380 nm, was recorded at 510 nm using a photomultiplier tube, a photon counter and an IBM microcomputer (as above). An extracellular calibration curve was constructed by measuring decreases in the intensity of fluorescence produced with increasing [Mg2+] in a solution containing 130 mM-[KCI], 10 mM-[HEPES] and 0.1 PM-[magfura] (non-esterified). It has not proved possible to quantify basal [Mg2+]i using magfura/AM, probably due to different Kds for the Mg2+- magfura complex in the intra- and extracellular environments (111. Thus, for the purpose of this study, it was assumed that this value was 0.5 mM, similar to that found in other cell types (12). The extent of any change in [Mg2+]i following a given treatment was assessed by measuring the fall in intracellular fluorescence at 380 nm and relating this to the calibration curve.
RESULTS Effect of deuolarisation experiments
examined
on [Ca2+]i
responses due to elevated [Ca2+],
the effect of depolarisation
response due to 20 mM-[Ca2+],
(Fieure
1):
The first set of
with 100 mM-IItx& L . 7..,.
500-
[Ca”]
: i
400-
‘.,
:.
3005 +“m 0
-
200-
loo50-
40 set FIGURE 1 The effect of depolarisation, by exposure to 100 mM-[K+], upon the elevation of intracellular free calcium levels ([Ca2+]t) (nM) in response to elevated extracellular calcium levels (]Ca2+1& (rnM). The steps represent changes in extracellular [Ca2+] or [K+]. Top panel: [Ca2+l - 1.25 mM (low), 20 mM (high); [K+] 5 mM (low), 100 mM (high). Bottom panel: [Ca2+] - 1.25 mM (low), 5 mM (high); [K+l - 5 mM (low), 100 mM (high).
application
of 5 mM-[Ca2+le
5) elevation
of [Ca2+]i.
of [Ca2+]i
Effect
was again
When observed
of hvwrpolarisation
experiments,
[Caz+],
response.
We found In another
presence
and absence elevation
PM-[valinomycin]
the [K+]
on [Ca2+]i
that
of 100 mM-IK+]
was returned
on application
was maintained
[Ca2+],
0.006)
in the presence
at 5 mM
valinomycin
which
did not significantly
due
elevated
to elevated
the plateau
the magnitude were
returned
a 2.15 k 0.31 fold (-+ S.E.M.;
a 4.35 f 1.32 fold
and valinomycin
of 1 PM-[valinomycin]
of [Ca2+Ji
to 5 mM,
only
(+ S.E.M.;
n =
n = 5) elevation
of 5 mM-[Ca2+],
responses
set of experiments,
produced
to basal
elevate
added phase
of [Ca2+]i
compared. following
[Ca2+]i 621
([Ca2+]i
[Ca2+],
(Figure
2):
at the plateau of the ]Ca2+k
responses
5 mM-[Ca2+], a wash ?I S.E.M.;
due
In the first phase
of the [Ca2+]i
response
due to 5 mM-
to 5 mM-[Ca2+],
produced with nM;
set of
BSS ([Ca2+le
in
a significant - 1.25 mM).
the (p = 1
61.3 + 4.03 and 81 f 12.3; p =
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
[Ca”]
I 1 VM-[valinomycin] .
5007
-_
_-
_.
_.-,,
,..
_.” ‘..
400300I c +=
200-
% 52.
1 oo-
._ . . .... ,.
50.,- .._.._._...._.
_
. . . . . . . . . . .._.......~
., ,...,...
. . . . . ::-
......’
..,
40 set
O-
I
[Ca*+] 1 PM-[valinomycin] -. $. .,.. ,:y...ATT .-.cI-.- :. .-
40 set
OJ
FIGURE 2 The effect of hyperpolarisation, by exposure to IPM-[valinomycin], upon the elevation of intracellular free calcium levels ([Ca2+]i) (nM) in response to elevated extracellular calcium levels ([Ca2+],J (mM). The steps represent changes in extracellular ]Ca2+] (]K+] - 5 mM). ]Ca2+1 - 1.25 mM (low), 5 mM thigh).
0.15; n = 4; with
and
without
mM-[Ca2+],
in the presence
mM-[Ca2+]i
was
isolated fold caused
experiments, 0.5 mM
affinity
an elevation
resulted
of valinomycin,
the [Ca2+]t
(p = 0.05)
without
the drug
greater
using for
of [Mg2+h
in a rise in [Mg2+]t the exposure to 0.85 + 0.03
compared without from
in the presence
(k S.E.M.;
(Figure
fluorochrome, with
elevating
an assumed
of osteoclasts mM
response
on [Mg2+]i
the Mg 2+-sensitive Mg2+
However,
when
osteoclasts
were
was potentiated.
Thus,
of valinomycin
fA[Ca2+]i
exposed
A[Ca2+]i
to 5
due to 5
t- S.E.M.;
nM;
(68.5 It: 9.63; n = 4).
of [Ca2+]&[Mg2+],elevation
osteoclasts
higher
respectively).
significantly
181 -t 29.5; n = 4) than
The effects
valinomycin,
Ca2+.
magfura.
The elevation
[Ca2+]t. level
3): We have
Thus,
of 0.5 mM
to 10 mM-]Mg2+leraised n = 4, p = 0.001).
622
The
measured dye
of [Mg2+],
increasing
]Mg2+ Subsequent
Ii
to between
fn = 2).
from exposure
signals
has approximately,
[Mg2+lefrom
to 1.19 mM
Mg2+i
a 30-
5 and
0.8 mM
of these
basal cells
40 mM
to 40 mM
In a separate
the assumed
in
set of level
of
to 5 @4-
Vol.
183,
No.
BIOCHEMICAL
2, 1992
,
60000
AND
BIOPHYSICAL
cooool-o' 0.5
0.0
0.5
0
1.0
10
1.5
20
2.0
1
RESEARCH
1.5
2.5
30
2
COMMUNICATIONS
Ws2*l
3.0
Ws2+l
40
[Mg”l,
16
a
b
C
FIGURE 3 Top panel: The effect of a range of concentrations (mM) of ionised magnesium ([Mg2+1) on the fluorescence of the Mg2+-magfura complex (expressed as photon counts per second). Excitation wavelength - 380 nm; emissions recorded at 510 nm. The insert shows a representative trace. Middle panel: The effect of a range of concentrations (mM) of ionised magnesium applied extracellularly (+ S.D.) (mM), calculated from the extracellular ([M$+]&, upon intracellular free Mg 2+ (IMg2+&t calibration curve (top panel) assuming a basal [Mg2+]i of 0.5 mM. Data derived from two experiments. Bottom panel: The effect of various treatments, applied sequentially to osteoclasts, upon intracellular free Mg2+ ([Mgz+]i) (mM). The values have been calculated from the extracellular calibration curve (top panel) assuming a basal [MgI+li of 0.5 mM. The stacked bar diagram represents levels before (hatched bars) and after (open bars) the following treatments: a - 10 mM-[Mg2+],: b - 10 mM-[Mg2+], + 5 PMFor statistics: see Results [valinomycin]; c - 10 mM-[Mg2+le + 5 PM-[valinomycin] + 20 mM-[Ca2+], section.
[valinomycin]
in the presence of 10 mM-[Mg2+],
produced a further elevation of [Mg’+]i
(rt S.E.M.; n = 6; p = 0.004). On the addition of 20 mM-[Ca2+],(in [valinomycin]),
there was a further rise in [M$+li
[Ca2+], was also capable of increasing [Mgz+]i
to 1.19 + 0.06 mM
the presence of 10 mM-[Mg2+fle and 5 vM-
to 1.42 + 0.15 mM (k S.E.M.; n = 4; p = 0.038). Raised even
in the absence of valinomycin
623
(not shown).
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
DISCUSSION It is presently ROCCs) actually allowed
controversial
exist (13).
for their prediction
as to whether
However,
or not receptor-operated
the results described
in a variety of eukaryotic
modulate Ca2+ influx.
term of this gradient,
the response. However,
characteristically
it should
depolarisation gradient
be noted
that unlike
explanation
may be modulated
physiological
their membrane
the basis for a potential
the magnitude
Ca*+
channels
It is known
of Ca*+ gradient.
(9), where
14). These studies raise the possibility
Whatever
voltage during
An
of the osteoclast stems
and hence, its sensitivity
implication.
mechanism
potentiates
reverse the effects of depolarisation.
voltage.
to changes in [Ca*+],
the explanation,
It has recently
this phenomenon
been demonstrated
that
resorptive activity (15). The results therefore
by which
[Ca*+]i, its sensitivity to changes in [Ca*+], and hence,
Hallam
significantly
of 1 mM, in this case the high concentration
partially
Ca *+ “receptor”
by changes in membrane
to have an important
the permeation
agonist-activated
in skeletal muscle (review:
of the putative
osteoclasts can manipulate provide
could
voltage, will
by the electrical term of the electrochemical other
(9).
attenuates the [Ca*+]i
for the observed effects of voltage on Ca *+-sensitivity
from studies on charge movements that the configuration
using 100 mM-[K+] with valinomycin
attenuate Ca *+ influx at a ]Ca*+],
would
Ca2+ influx
across the cell membrane
of the putative Ca*+ “receptor”,
modulated
set up by 5 or 20 mM-[Ca2+]e
alternative
appears
hyperpolarisation
Thus, in response to activation
influx is somewhat
gradient
for
have in the past,
caused by a change in membrane
We have found that depolarisation
response due to elevated [Ca*+]e whilst
lines, which
cells (9). Firstly, receptor-operated
has been found to be sensitive to changes in the electrochemical Hence, a change in the electrical
follow
calcium channels
membrane
the
physiological
voltage may modulate
osteoclast
function of these cells.
that ROCCs have a high degree of selectivity for divalent cations (16), and that
properties
of the respective cations follow a rank order of potency.
and Rink (1985) established
a method
for the quantification
of Mn*+
Using this concept,
influx
by utilising
the
ability of the cation to quench fluorescence emitted by the dye (17). Hem, we have developed the use of a Mg2+-sensitive
fluorochrome,
magfura,
to demonstrate
Mg*+
influx.
Magfura
has a 250-fold
lower
affinity for Ca*+ than does fura (18). However, at present, we can report only rough estimates of [Mg*+li changes as a rigorous
calibration
is fraught with technical difficulties.
We have found that Mg*+ and
Ca*+ are equally potent activators of Mg *+ influx. Thus sustained influx of Mg*+ follows [Ca*+],or(Mg*+],
providing
clear evidence that “receptor”
the elevation of
activation by Ca*+ can cause the permeation
of a different divalent cation, Mg *+. The results would argue for a fundamental
biophysical
the processes of cation-induced
although
“receptor”
activation
and cation permeation,
separation of “receptor”
and
channel may be two parts of a single structural entity.
Acknowledgments: The studies were supported in part by Arthritis and Rheumatism Council, U.K. (MZ), Leverhulme Trust, U.K. (MZ), Sandoz Foundation for Gerontological Research, Switzerland (MZ) and Medical Research Council, U.K. (MZ). We are grateful to Professor Iain MacIntyre for advice. 624
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1.
2. 3.
Silver, A., Murrils, R.J., and Etherington, D.J. (19881 Microelectrode studies on microenvironment beneath adjacent macrophages and osteoclasts. Exp. Cell Res. 175,266-276. Zaidi, M., Datta, H.K., Moonga, B.S., Patchell, A., and MacIntyre, I. (1989) Biochem. Biophys. Res. Commun. 163,1461-1465. Malgaroli, A., Meldolesi, J., Zambonin-Zallone, A., and Teti, A. (19891 J. Biol. Chem. 264, 1434214347.
4. 5. 6. 7. 8. 9. 10.
11.
12. 13. 14. 15. 16. 17. 18.
Zaidi, M. (1990) Biosc. Rep. 10,493-507. Moonga, B.S., Moss, D.W., Patchell, A., and Zaidi, M. (19901 J. Physiol. 490, 29-46. Zaidi, M., Kerby, J., Huang, C.L.-H., Alam, A.S.M.T., Rathod, H., Chambers, T.J., and Moonga, B.S. (1991) J. Cell Physiol. 187, 472-478. Datta, H.K., Maclntyre, I., and Zaidi, M. (1990) Biochem. Biophys. Res. Commun. 167, 183-188. Brown, E.M. (19911 Physiol. Rev. 71, 371-411. Benham, CD., Meritt, J.E., and Rink, T.J. (1989) In: Ion Transport. Eds. Keeling, D., Benham, CD. Academic Press, London. pp 197-213. Zaidi, M., Alam, A.S.M.T., Bax, C., Shankar, V., Bevis, P.J.R., Huang, C.L.H., and Moonga, B.S. (1991) In: Methods in Molecular Biology, Volume: 2, Membrane Methods. Ed: Graham, J.M. Humana Press Inc. (New Jersey), In the press. Murphy, E., Freudenrich, C.C., Levy, L.A., London R.E., and Lieberman, M. (1989) Proc. Natl. Acad. Sci. USA 86,2981-2984. Roe, M.W., Lemasters, J.J.,and Herman, B. (1990) Cell Calcium 11, 63-73. Rink, T.J. (1988) Nature 334, 649-650. Huang, C.L.-H. (1988) Physiol. Rev. 68, 1197-1247. Arkett, S.A., Dixon, SJ., and Sims, S.M. (1991) J. Bone Min. Res. (abstract) 6,51. Zschauer, A., van Breenan, C., Buhler, F.R., and Nelson, M.T. (1988) Nature 334, 703-705. Halam, T.J., and Rink, T.J. (19851 FEBS Lctt. 186, 175-179. Raju, B., Murphy, E., Levy, L., and London R.E. (1989) Am. J. Physiol. 256, C540-C548.
625
183,
March
No.
2, 1992
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
Paqes
16, 1992
IS THE
OSTEOCLAST
CALCIUM
C.M.R.
Bax, V. SShankar,
Department
*The
Received
of Cellular
Physiological
January
Elevated calcium
B. S. Moonga,
C. L.-H.
Huang*,
Sciences, St. George’s SW17 ORB , U .K.
University
CALCIUM
of Cambridge,
and
CHANNEL
?
M. Zaidi
Hospital
Medical
Cambridge
School,
CB2 3EG,
U . K.
1992
calcium levels presumably
whether or not Ca2+-induced a divalent cation channel. a distinct
A RECEPTOR-OPERATED
and Molecular London
Laboratory,
20,
extracellular levels ([Ca2+]i),
comprises
“RECEPTOR’
619-625
([Ca2+],) inhibit via the activation
cytosolic
free
It is unclear involves the direct gating, by the putative “receptor”, of that ICa2+]i elevation in response to elevated [Ca2+],
[Ca2+]i elevation The results show
component
osteoclast function by elevating of a surface Ca2+ “receptor”.
of Ca 2+ influx,
the magnitude
of which
can be decreased
and
increased,
respectively, by depolarising (100 mM-[K+]) and hyperpolarising (1 PM-[valinomycin]) the osteoclast membrane. In addition, activation of the putative Ca2+ “receptor” by elevated [Ca2+]ecauses influx of the related divalent cation, magnesium (Mg2+). We suggest that Ca2+ influx induced by Ca2+ “receptor” activation is a major component of the observed [Ca2+]i response. ~ 194.2 AC.3&rnlC Pm2SL. IIIC.
During high
the process
concentrations
the cell (1). directly
We have
regulate Ca2+
accompanied
with (3,4),
into
osteoclast
Ca2+
chief
cells
“receptor” other
divalent
receptors would
lowered
of depolarisation examined Mg2+-sensitive
the ambient (2).
release Ca2+
a rise of [CaZ+]i
cations.
upon and
the effects
that
present would
shown
is determined its membrane
cell retraction
bone
resorption
detects
a change
that
to that found explored
potential
of Ca2+
the influx
(review, upon
extracellular
magfura.
[Ca2+],induccd
(7).
of Ca2+,
induced
gradient
across therefore
[Ca2+]i ([Mg2+]3
In these
clear that
respects,
in response
attcmptcd
Ca2+
by Caz+
to stimulation
the cell membrane
the
secreting
putative
to its activation
elevation
it then
hormone the
3)
cell-matrix
(4, 6) which
or not
elevation.
(2,
becoming
on parathyroid
in response
9). We have
magnesium
(4), reduced
in [Ca2+],
site can
([Ca2+]i)
It is now
whether
of
to an elevated
Ca2+
mechanism
have
the influx
(2).
to
border
resorptive
osteoclasts
(2), including
by the electrochemical
hyperpolarisation of [Ca2+]eand
allow
rat
exposed
the ruffled
at the osteoclast
of isolated
is similar we
beneath
of cytosolic
or “receptor”
study,
and is consequently
an elevation
(5) and reduced
system
It has been
fluorochrome,
concentration
exposure
inhibition
generates
rise up to 30 mM
by a dihydropyridinc-insensitive
In the
a channel
Ca2+ The
sensor
detection-transduction 8).
may
causes
of functional
enzyme
the osteoclast these
([Ca*+]3
a unique
by agonists, depend
that
resorption, (Ca2+);
function
evidence
(review: gates
shown
concentration
in the rat osteoclast, transduces
calcium
osteoclast
extracellular
adhesion
of bone
of ionised
or of
and hence,
to study
the effects
In addition,
we have
on Mg2+
influx
using
the
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Osteoclast isolation and culture: Femora and tibiae obtained from neonatal rats were cleaned and cut across their epiphyses in N-2-hydroxyethyl piperazine-N’-2-ethane sulphonic acid (HEPESl-buffered Medium 199 supplemented with foetal calf serum (FCS) (10% v/v) tall from ICN Flow, UK). Osteoclasts were disaggregated by curetting bones into medium and agitating the suspension with a pipette. The supematant was then dispersed onto 22 mm glass coverslips (0 grade) and cells were allowed to sediment and attach for 30 minutes at 3X. Measurement
of [Ca2+]ii Osteoclasts, settled onto glass coverslips, were incubated with the ester of fura 2 (fura 2/AMl (10 PM) (Novobiochem, U.K.1 in Medium 199 for 40 minutes. The coverslips with the dye-loaded cells were then transferred to a perspex bath mounted onto the stage of a microspectrofluorimeter constructed from an inverted microscope (Diaphot; Nikon, Telford, U.K.). Osteoclasts, constantly bathed in buffered salt solution (BSS) containing 130 mM-[NaCl], 5 mM-[KCl], 1.25 mM-[CaCl2], 0.8 mM-[MgCl2], 5 mM-[glucose] and 10 mM-[HEPES] (pH - 7.4), were exposed alternately to excitation wavelengths of 340 and 380 nm using a computer-controlled spinning filter wheel. The fluorescent signal was directed to the sideport of the microscope which contained a 510 nm interference filter. The emission, obtained from single osteoclasts at the two excitation wavelengths, was fed into a photomultiplier tube (PM28B, Thorn EMI, Middlesex, U.K.). Single photon currents were then counted by a photon counter (Newcastle Photometric Systems, U.K.). Photons s-1 in each channel were recorded in an IBM microcomputer and the ratio of excitation intensities fF340/F380) calculated and displayed, [Ca2+]i was determined from a standard curve obtained by intracellular calibration (10). acetomethoxy
Measurement of [Mg2+]i : Osteoclasts, settled onto glass coverslips, were incubated with the acetomethoxy ester of magfura (magfura/AM) (1 l.rMl (Novobiochem, U.K.1 in Medium 199 for 30 minutes. The coverslips with the dye-loaded cells were then transferred onto the stage of a microspectrofluorimeter and incubated as described above. The emission intensity, following excitation of the cells at 380 nm, was recorded at 510 nm using a photomultiplier tube, a photon counter and an IBM microcomputer (as above). An extracellular calibration curve was constructed by measuring decreases in the intensity of fluorescence produced with increasing [Mg2+] in a solution containing 130 mM-[KCI], 10 mM-[HEPES] and 0.1 PM-[magfura] (non-esterified). It has not proved possible to quantify basal [Mg2+]i using magfura/AM, probably due to different Kds for the Mg2+- magfura complex in the intra- and extracellular environments (111. Thus, for the purpose of this study, it was assumed that this value was 0.5 mM, similar to that found in other cell types (12). The extent of any change in [Mg2+]i following a given treatment was assessed by measuring the fall in intracellular fluorescence at 380 nm and relating this to the calibration curve.
RESULTS Effect of deuolarisation experiments
examined
on [Ca2+]i
responses due to elevated [Ca2+],
the effect of depolarisation
response due to 20 mM-[Ca2+],
(Fieure
1):
The first set of
with 100 mM-IItx& L . 7..,.
500-
[Ca”]
: i
400-
‘.,
:.
3005 +“m 0
-
200-
loo50-
40 set FIGURE 1 The effect of depolarisation, by exposure to 100 mM-[K+], upon the elevation of intracellular free calcium levels ([Ca2+]t) (nM) in response to elevated extracellular calcium levels (]Ca2+1& (rnM). The steps represent changes in extracellular [Ca2+] or [K+]. Top panel: [Ca2+l - 1.25 mM (low), 20 mM (high); [K+] 5 mM (low), 100 mM (high). Bottom panel: [Ca2+] - 1.25 mM (low), 5 mM (high); [K+l - 5 mM (low), 100 mM (high).
application
of 5 mM-[Ca2+le
5) elevation
of [Ca2+]i.
of [Ca2+]i
Effect
was again
When observed
of hvwrpolarisation
experiments,
[Caz+],
response.
We found In another
presence
and absence elevation
PM-[valinomycin]
the [K+]
on [Ca2+]i
that
of 100 mM-IK+]
was returned
on application
was maintained
[Ca2+],
0.006)
in the presence
at 5 mM
valinomycin
which
did not significantly
due
elevated
to elevated
the plateau
the magnitude were
returned
a 2.15 k 0.31 fold (-+ S.E.M.;
a 4.35 f 1.32 fold
and valinomycin
of 1 PM-[valinomycin]
of [Ca2+Ji
to 5 mM,
only
(+ S.E.M.;
n =
n = 5) elevation
of 5 mM-[Ca2+],
responses
set of experiments,
produced
to basal
elevate
added phase
of [Ca2+]i
compared. following
[Ca2+]i 621
([Ca2+]i
[Ca2+],
(Figure
2):
at the plateau of the ]Ca2+k
responses
5 mM-[Ca2+], a wash ?I S.E.M.;
due
In the first phase
of the [Ca2+]i
response
due to 5 mM-
to 5 mM-[Ca2+],
produced with nM;
set of
BSS ([Ca2+le
in
a significant - 1.25 mM).
the (p = 1
61.3 + 4.03 and 81 f 12.3; p =
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[Ca”]
I 1 VM-[valinomycin] .
5007
-_
_-
_.
_.-,,
,..
_.” ‘..
400300I c +=
200-
% 52.
1 oo-
._ . . .... ,.
50.,- .._.._._...._.
_
. . . . . . . . . . .._.......~
., ,...,...
. . . . . ::-
......’
..,
40 set
O-
I
[Ca*+] 1 PM-[valinomycin] -. $. .,.. ,:y...ATT .-.cI-.- :. .-
40 set
OJ
FIGURE 2 The effect of hyperpolarisation, by exposure to IPM-[valinomycin], upon the elevation of intracellular free calcium levels ([Ca2+]i) (nM) in response to elevated extracellular calcium levels ([Ca2+],J (mM). The steps represent changes in extracellular ]Ca2+] (]K+] - 5 mM). ]Ca2+1 - 1.25 mM (low), 5 mM thigh).
0.15; n = 4; with
and
without
mM-[Ca2+],
in the presence
mM-[Ca2+]i
was
isolated fold caused
experiments, 0.5 mM
affinity
an elevation
resulted
of valinomycin,
the [Ca2+]t
(p = 0.05)
without
the drug
greater
using for
of [Mg2+h
in a rise in [Mg2+]t the exposure to 0.85 + 0.03
compared without from
in the presence
(k S.E.M.;
(Figure
fluorochrome, with
elevating
an assumed
of osteoclasts mM
response
on [Mg2+]i
the Mg 2+-sensitive Mg2+
However,
when
osteoclasts
were
was potentiated.
Thus,
of valinomycin
fA[Ca2+]i
exposed
A[Ca2+]i
to 5
due to 5
t- S.E.M.;
nM;
(68.5 It: 9.63; n = 4).
of [Ca2+]&[Mg2+],elevation
osteoclasts
higher
respectively).
significantly
181 -t 29.5; n = 4) than
The effects
valinomycin,
Ca2+.
magfura.
The elevation
[Ca2+]t. level
3): We have
Thus,
of 0.5 mM
to 10 mM-]Mg2+leraised n = 4, p = 0.001).
622
The
measured dye
of [Mg2+],
increasing
]Mg2+ Subsequent
Ii
to between
fn = 2).
from exposure
signals
has approximately,
[Mg2+lefrom
to 1.19 mM
Mg2+i
a 30-
5 and
0.8 mM
of these
basal cells
40 mM
to 40 mM
In a separate
the assumed
in
set of level
of
to 5 @4-
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,
60000
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BIOPHYSICAL
cooool-o' 0.5
0.0
0.5
0
1.0
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1.5
20
2.0
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RESEARCH
1.5
2.5
30
2
COMMUNICATIONS
Ws2*l
3.0
Ws2+l
40
[Mg”l,
16
a
b
C
FIGURE 3 Top panel: The effect of a range of concentrations (mM) of ionised magnesium ([Mg2+1) on the fluorescence of the Mg2+-magfura complex (expressed as photon counts per second). Excitation wavelength - 380 nm; emissions recorded at 510 nm. The insert shows a representative trace. Middle panel: The effect of a range of concentrations (mM) of ionised magnesium applied extracellularly (+ S.D.) (mM), calculated from the extracellular ([M$+]&, upon intracellular free Mg 2+ (IMg2+&t calibration curve (top panel) assuming a basal [Mg2+]i of 0.5 mM. Data derived from two experiments. Bottom panel: The effect of various treatments, applied sequentially to osteoclasts, upon intracellular free Mg2+ ([Mgz+]i) (mM). The values have been calculated from the extracellular calibration curve (top panel) assuming a basal [MgI+li of 0.5 mM. The stacked bar diagram represents levels before (hatched bars) and after (open bars) the following treatments: a - 10 mM-[Mg2+],: b - 10 mM-[Mg2+], + 5 PMFor statistics: see Results [valinomycin]; c - 10 mM-[Mg2+le + 5 PM-[valinomycin] + 20 mM-[Ca2+], section.
[valinomycin]
in the presence of 10 mM-[Mg2+],
produced a further elevation of [Mg’+]i
(rt S.E.M.; n = 6; p = 0.004). On the addition of 20 mM-[Ca2+],(in [valinomycin]),
there was a further rise in [M$+li
[Ca2+], was also capable of increasing [Mgz+]i
to 1.19 + 0.06 mM
the presence of 10 mM-[Mg2+fle and 5 vM-
to 1.42 + 0.15 mM (k S.E.M.; n = 4; p = 0.038). Raised even
in the absence of valinomycin
623
(not shown).
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DISCUSSION It is presently ROCCs) actually allowed
controversial
exist (13).
for their prediction
as to whether
However,
or not receptor-operated
the results described
in a variety of eukaryotic
modulate Ca2+ influx.
term of this gradient,
the response. However,
characteristically
it should
depolarisation gradient
be noted
that unlike
explanation
may be modulated
physiological
their membrane
the basis for a potential
the magnitude
Ca*+
channels
It is known
of Ca*+ gradient.
(9), where
14). These studies raise the possibility
Whatever
voltage during
An
of the osteoclast stems
and hence, its sensitivity
implication.
mechanism
potentiates
reverse the effects of depolarisation.
voltage.
to changes in [Ca*+],
the explanation,
It has recently
this phenomenon
been demonstrated
that
resorptive activity (15). The results therefore
by which
[Ca*+]i, its sensitivity to changes in [Ca*+], and hence,
Hallam
significantly
of 1 mM, in this case the high concentration
partially
Ca *+ “receptor”
by changes in membrane
to have an important
the permeation
agonist-activated
in skeletal muscle (review:
of the putative
osteoclasts can manipulate provide
could
voltage, will
by the electrical term of the electrochemical other
(9).
attenuates the [Ca*+]i
for the observed effects of voltage on Ca *+-sensitivity
from studies on charge movements that the configuration
using 100 mM-[K+] with valinomycin
attenuate Ca *+ influx at a ]Ca*+],
would
Ca2+ influx
across the cell membrane
of the putative Ca*+ “receptor”,
modulated
set up by 5 or 20 mM-[Ca2+]e
alternative
appears
hyperpolarisation
Thus, in response to activation
influx is somewhat
gradient
for
have in the past,
caused by a change in membrane
We have found that depolarisation
response due to elevated [Ca*+]e whilst
lines, which
cells (9). Firstly, receptor-operated
has been found to be sensitive to changes in the electrochemical Hence, a change in the electrical
follow
calcium channels
membrane
the
physiological
voltage may modulate
osteoclast
function of these cells.
that ROCCs have a high degree of selectivity for divalent cations (16), and that
properties
of the respective cations follow a rank order of potency.
and Rink (1985) established
a method
for the quantification
of Mn*+
Using this concept,
influx
by utilising
the
ability of the cation to quench fluorescence emitted by the dye (17). Hem, we have developed the use of a Mg2+-sensitive
fluorochrome,
magfura,
to demonstrate
Mg*+
influx.
Magfura
has a 250-fold
lower
affinity for Ca*+ than does fura (18). However, at present, we can report only rough estimates of [Mg*+li changes as a rigorous
calibration
is fraught with technical difficulties.
We have found that Mg*+ and
Ca*+ are equally potent activators of Mg *+ influx. Thus sustained influx of Mg*+ follows [Ca*+],or(Mg*+],
providing
clear evidence that “receptor”
the elevation of
activation by Ca*+ can cause the permeation
of a different divalent cation, Mg *+. The results would argue for a fundamental
biophysical
the processes of cation-induced
although
“receptor”
activation
and cation permeation,
separation of “receptor”
and
channel may be two parts of a single structural entity.
Acknowledgments: The studies were supported in part by Arthritis and Rheumatism Council, U.K. (MZ), Leverhulme Trust, U.K. (MZ), Sandoz Foundation for Gerontological Research, Switzerland (MZ) and Medical Research Council, U.K. (MZ). We are grateful to Professor Iain MacIntyre for advice. 624
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REFERENCES 1.
2. 3.
Silver, A., Murrils, R.J., and Etherington, D.J. (19881 Microelectrode studies on microenvironment beneath adjacent macrophages and osteoclasts. Exp. Cell Res. 175,266-276. Zaidi, M., Datta, H.K., Moonga, B.S., Patchell, A., and MacIntyre, I. (1989) Biochem. Biophys. Res. Commun. 163,1461-1465. Malgaroli, A., Meldolesi, J., Zambonin-Zallone, A., and Teti, A. (19891 J. Biol. Chem. 264, 1434214347.
4. 5. 6. 7. 8. 9. 10.
11.
12. 13. 14. 15. 16. 17. 18.
Zaidi, M. (1990) Biosc. Rep. 10,493-507. Moonga, B.S., Moss, D.W., Patchell, A., and Zaidi, M. (19901 J. Physiol. 490, 29-46. Zaidi, M., Kerby, J., Huang, C.L.-H., Alam, A.S.M.T., Rathod, H., Chambers, T.J., and Moonga, B.S. (1991) J. Cell Physiol. 187, 472-478. Datta, H.K., Maclntyre, I., and Zaidi, M. (1990) Biochem. Biophys. Res. Commun. 167, 183-188. Brown, E.M. (19911 Physiol. Rev. 71, 371-411. Benham, CD., Meritt, J.E., and Rink, T.J. (1989) In: Ion Transport. Eds. Keeling, D., Benham, CD. Academic Press, London. pp 197-213. Zaidi, M., Alam, A.S.M.T., Bax, C., Shankar, V., Bevis, P.J.R., Huang, C.L.H., and Moonga, B.S. (1991) In: Methods in Molecular Biology, Volume: 2, Membrane Methods. Ed: Graham, J.M. Humana Press Inc. (New Jersey), In the press. Murphy, E., Freudenrich, C.C., Levy, L.A., London R.E., and Lieberman, M. (1989) Proc. Natl. Acad. Sci. USA 86,2981-2984. Roe, M.W., Lemasters, J.J.,and Herman, B. (1990) Cell Calcium 11, 63-73. Rink, T.J. (1988) Nature 334, 649-650. Huang, C.L.-H. (1988) Physiol. Rev. 68, 1197-1247. Arkett, S.A., Dixon, SJ., and Sims, S.M. (1991) J. Bone Min. Res. (abstract) 6,51. Zschauer, A., van Breenan, C., Buhler, F.R., and Nelson, M.T. (1988) Nature 334, 703-705. Halam, T.J., and Rink, T.J. (19851 FEBS Lctt. 186, 175-179. Raju, B., Murphy, E., Levy, L., and London R.E. (1989) Am. J. Physiol. 256, C540-C548.
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