(Received for publication, July 21, 1994; and in revised form, October 20, 1994)
From the
Osteoclasts resorb bone by secreting protons into an
extracellular resorption zone through vacuolar-type proton pumps
located in the ruffled border. The present study was undertaken to
evaluate whether proton pumps also contribute to intracellular pH
(pH) regulation. Fluorescence imaging and
photometry, and electrophysiological methods were used to characterize
the mechanisms of pH regulation in isolated rabbit osteoclasts. The
fluorescence of single osteoclasts cultured on glass coverslips and
loaded with a pH-sensitive indicator was measured in nominally
HCO
-free solutions. When suspended in
Na
-rich medium, the cells recovered from an acute acid
load primarily by means of an amiloride-sensitive
Na
/H
antiporter. However, rapid
recovery was also observed in Na
-free medium when
K
was used as the substitute. Bafilomycin-sensitive,
vacuolar-type pumps were found to contribute marginally to pH
regulation and no evidence was found for
K
/H
exchange. In contrast,
pH
recovery in high K
medium was
largely attributed to a Zn
-sensitive proton
conductive pathway. The properties of this conductance were analyzed by
patch-clamping osteoclasts in the whole-cell configuration.
Depolarizing pulses induced a slowly developing outward current and a
concomitant cytosolic alkalinization. Determination of the reversal
potential during ion substitution experiments indicated that the
current was due to H
(equivalent) translocation across
the membrane. The H
current was greatly stimulated by
reducing pH
, consistent with a homeostatic role of
the conductive pathway during intracellular acidosis. These results
suggest that vacuolar-type proton pumps contribute minimally to the
recovery of cytoplasmic pH from intracellular acid loads. Instead, the
data indicate the presence of a pH- and membrane potential-sensitive
H
conductance in the plasma membrane of osteoclasts.
This conductance may contribute to translocation of charges and acid
equivalents during bone resorption and/or generation of reactive oxygen
intermediates by osteoclasts.
Osteoclasts are multinucleated cells which are primarily
responsible for bone resorption. These cells are located on the bone
surface and effect resorption of both the inorganic and organic
components of bone in a localized area underlying their attachment
site. Several lines of evidence suggest that acidification of the
osteoclast-bone interface plays a critical role in the resorptive
process. First, the pH of the localized extracellular compartment has
been determined to be acidic by fluorescence measurements as well as by
studies using microelectrodes (Baron et al., 1985; Silver et al., 1988). Second, low pH is capable of dissolving bone
mineral (hydroxyapatite) by a process whereby 2H exchange for every Ca
ion released from the
crystal (Neuman and Neuman, 1958). Finally, pharmacological agents
known to impair H
efflux from osteoclasts are able to
inhibit bone resorption (see below).
The primary cellular mechanism
responsible for the localized acidification of the osteoclast-bone
interface is a plasmalemmal vacuolar-type HATPase
(V-ATPase), (
)which actively extrudes protons into the
extracellular space. Following cell activation, osteoclasts attached to
the bone matrix create an underlying resorption zone characterized by a
highly folded membrane called the ruffled border, surrounded by an area
rich in actin (Kallio et al., 1971). Immunohistochemical
studies have documented the accumulation of V-ATPases within this
subdomain of the plasma membrane, allowing vectorial proton secretion
to the site of bone resorption (Blair et al., 1989;
Väänänen et al., 1990). Functional studies support this concept.
Bafilomycin A
, a specific V-ATPase blocker, inhibits bone
resorption (Bowman et al., 1988; Sundquist et al.,
1990), as does acetazolamide, an inhibitor of carbonic anhydrase (Hall
and Kenny, 1987; Hunter et al., 1991). The latter enzyme
facilitates the hydration of carbon dioxide, thereby generating
carbonic acid which is the primary source of protons pumped into the
resorption lacuna by the V-ATPase (Laitala and
Väänänen,
1993; Asotra et al., 1994).
Metabolic acidosis is known to
be associated with negative calcium balance and bone loss. Indeed,
acidification of the extracellular culture medium has been shown to
increase the bone resorption activity of osteoclasts cultured on bone
(Walsh et al., 1990; Goldhaber and Rabadija, 1987; Shibutani
and Heersche, 1993). While the mechanism underlying this effect is not
clear, such observations highlight the need to maintain cytoplasmic pH
(pH) homeostasis, since conditions leading to a pH
imbalance impair normal osteoclast function. Bicarbonate/chloride
(HCO
/Cl
) exchange has
been shown to counteract the intracellular alkalinization that tends to
develop during proton secretion into the resorption lacuna (Teti et
al., 1989). A Na
/H
antiporter
has also been reported to contribute to pH
regulation in these cells (Redhead and Baker, 1988; Hall and
Chambers, 1990). To our knowledge, these are the only pH regulatory
systems reported to operate in osteoclasts.
In certain cell types,
mechanisms other than the cation and anion exchangers have been
recently found to contribute importantly to pH homeostasis. Cotransporters, pumps and conductive pathways
can also translocate H
equivalents and can thereby
modulate pH
. The purpose of the present studies
was to investigate whether additional mechanisms of pH
regulation exist in mammalian osteoclasts. Given the
problems inherent to acquiring pure populations of osteoclasts, we used
single cell fluorescence techniques to examine pH
recovery in individual osteoclasts, identified
morphologically and using histochemistry. These studies confirmed the
role of the Na
/H
antiporter as a
major pH
regulatory mechanism in acid-loaded
osteoclasts but also indicated that the plasmalemmal V-ATPase, while
crucial to bone resorption, plays a minor role in pH
homeostasis. Additionally, combined single-cell fluorescence and
patch-clamp techniques revealed the presence of a proton-conductive
pathway, which contributed significantly to pH
regulation, particularly when the
Na
/H
antiporter was rendered
inoperative.
To
load the osteoclasts with acid, adherent cells were incubated in
Hepes-RPMI containing 40 mM NHCl for 12 min at 37
°C and then rapidly transferred to a
NH
-free Na
- or
K
-containing medium. Where indicated, this technique
was used repeatedly to examine pH
recovery under different
conditions in a single cell.
Cellular buffering power was measured by pulsing control or acid-loaded cells with ammonium and calculated as described (Roos and Boron, 1981).
Currents in response to voltage steps were filtered
at 100 Hz with a 4-pole Bessel filter and digitized on line at 3-ms
intervals (unless indicated otherwise) using pClamp Clampex software
(Axon Instruments Inc.). Data analysis was carried out using pClamp
Clampfit and Clampan (Axon Instruments Inc.) and Sigma Plot (Jandel
Scientific) software. Where indicated, traces were normalized relative
to the maximal current generated by depolarizing the cell to +75
mV. These relative current values facilitate comparison between
experiments. Leak current was determined by stepping the voltage from
the holding potential of -60 mV to -90 mV in two 15-mV
steps. When present, significant linear leak was subtracted from the
current traces prior to analysis. All current records are shown with
the convention that an upward deflection indicates outward current
flow. ``Steady state'' currents were quantified over the last
100 ms of the 2.9-s pulse. Tail current amplitudes were determined by
curve fitting and back-extrapolating to the first time point of the
test pulse. All patch clamping experiments were carried out at room
temperature (22 °C). Data are presented as representative
traces of at least three similar experiments with cells from different
animals or as means ± standard error (S.E.) of the number of
experiments indicated. The capacitance of the cells was found to range
from 35 to 80 pF (mean ± S.E. were 55.9 ± 5.1 pF).
When electrophysiological and fluorescence data were collected
simultaneously, osteoclasts were loaded with BCECF by adding the free
acid of BCECF (300 µM) directly to the pipette solution.
These experiments were carried out at room temperature (22 °C).
Figure 1:
Phenotypic characterization and
pHrecovery of osteoclasts cultured on glass
coverslips. Upperpanel, light micrographs of
TRAP-stained osteoclasts illustrating the different morphological
types: A, giant spread osteoclast; B and C, motile osteoclasts of intermediate size; D and E, compact osteoclasts. The bar (panel A) represents
40 µm and applies to A-E. Lower panel,
pH
recovery of acid-loaded osteoclasts.
Osteoclasts were loaded with BCECF-AM and incubated with 40 mM NH
Cl for 20 min at 37 °C. Next, the cells were
washed and resuspended in K
medium at 37 °C and
fluorescence ratio images were collected as described under
``Experimental Procedures'' at 0, 20, 140, and 260 s after
the solution change (top row). The same cell was then reloaded
with NH
Cl and recovery in Na
medium
monitored. Reversing the order of the recovery solutions (i.e. Na
solutions after the first acid load and
K
after the second) yielded comparable results. Upon
completion of the experiment, the pH of the cell was set to 6.3, 6.8,
and 7.3 using nigericin/K
and the resulting images
along with the pH pseudocolor scale are illustrated to the left of the main panel. Data are representative of 3 similar
experiments.
As illustrated in Fig. 1(lowerpanel), the cytosol of
osteoclasts acidified uniformly following an
NH prepulse. In cells bathed in
Na
-rich medium a rapid recovery of pH
ensued, with restoration of the base-line level within
4 min.
A more gradual recovery was noted in Na
-free,
K
-rich solution (Fig. 1). No subcellular
pH
gradients were found to develop during the recovery from
acid loading in either solution at any of the times tested. The
mechanisms underlying H
extrusion in Na
and K
-rich media are analyzed in detail below. A
photomultiplier-based fluorescence ratio system, rather than imaging,
was used for the subsequent experiments to improve the temporal
resolution while minimizing photodynamic damage of the cells.
Figure 2:
pH recovery of
acid-loaded osteoclasts. Osteoclasts were loaded with BCECF-AM and
incubated with 40 mM NH
Cl for 20 min at 37 °C.
The pH
of single cells was measured
photometrically. A, data collection was started upon
superfusion with Na
medium (without NH
Cl)
at 37 °C (open circles; n = 4). The cell
was then reloaded with NH
Cl and recovery from the acid load
was monitored in Na
medium containing 1 mM amiloride (solidcircles; n =
5). Data are means ± S.E. of the indicated number of
experiments. B, acid-loaded osteoclasts were superfused in
K
medium (opencircles) or
NMG
medium (solidcircles) at 37
°C. Data are means ± S.E. of
5 similar
experiments.
As an
alternative test of this hypothesis, recovery from an acid load was
studied in media devoid of Na. As shown in Fig. 2B, replacement of Na
by the
purportedly impermeant NMG
yielded results that were
comparable to those obtained with amiloride, i.e. a profound
acidification followed by an insignificant recovery. Unexpectedly, a
sizable recovery (0.35 ± 0.13 pH min; n = 6) was
noted in K
-rich medium devoid of Na
(opencircles). Because osteoclasts are known
to display a prominent K
conductance (Arkett et
al., 1992), it is likely that the cells depolarized in
K
solution. These data suggest that the membrane
potential may be an important determinant of the pH
recovery observed in Na
-free medium. The
Na
-independent pH
regulatory processes
were studied in detail below.
Figure 3:
Effects of bafilomycin A and
of Zn
on pH
recovery in
K
medium. Left panel, osteoclasts were loaded
with BCECF-AM and then incubated with 40 mM NH
Cl
for 20 min at 37 °C. The cells were next superfused with
K
medium containing 100 µM Zn
(solid circles). Alternatively, the cells were
pretreated with 200 nM bafilomycin A
during the
final 5 min of the acid-loading step and then exposed to K
medium containing the same concentration of bafilomycin (open
circles). Traces are means ± S.E. of
5 similar
experiments. Right panel, acridine orange staining of a
control cell (upper images) or a cell treated for 5 min with
200 nM bafilomycin A
(lowerimages). The orange and green components of the
fluorescent emission from the same cell were separated by use of
appropriate filters and are displayed separately. Orange fluorescence,
indicative of acidic organelles, is shown in the leftimages, and green fluorescence, reflective of dye
distributed at near-neutral pH, is shown to the right.
Figure 4:
Outward-rectifying, slowly activating,
Zn-sensitive current. Osteoclasts were patched in the
whole-cell configuration with a pipette containing NMG-Asp 6.0. The
holding potential was clamped at -60 mV, and pulses lasting 2.9 s
were applied between -90 and +75 mV in 15-mV increments at
30-s intervals. A, family of currents from a 39-pF osteoclast
patched with a 7-M
pipette. The bath was perfused with NMG-Asp pH
7.5. B, patched osteoclasts were perfused with NMG-Asp pH 7.5
solution (solid circles) and a family of current traces like
those in A were generated. The bath was next perfused
with solution NMG-Asp/Zn
pH 7.5 (opentriangles), and another family of currents was recorded.
Finally, the bath was perfused with the original
Zn
-free solution (solidtriangles),
and one last current family generated. The magnitude of the current was
averaged over the last 150 ms of each voltage pulse. To facilitate
comparison between experiments, the average current determined during
the +75-mV pulse from the first family of traces was set to 1 and
all other currents from each cell are expressed relative to this value.
The relative currents of four cells from different animals are plotted versus the applied potential. Steady-state currents prior to
application of test pulses were negligible. Values are means ±
S.E. (n = 4). Where absent, bars were smaller than the
symbols.
While the conditions used
above facilitate detection of the conductance, they are unphysiological
(low pH and cytosolic media devoid of K
).
However, the outward current can also be readily elicited at near
physiological pH
(6.9) and intracellular K
concentrations (solution K-Asp 6.9; Table 1). In fact,
these conditions were used below to assess the effects of K
channel antagonists.
Figure 5:
Depolarization increases pH and activates an outward current. BCECF-loaded osteoclasts
on glass coverslips were patched in the whole-cell configuration at a
holding potential of -60 mV. The patch pipette contained NMG-Asp
low
6.5, and the bath was continually perfused with solution
NMG-Asp 7.5. Cells were then subjected to voltage steps lasting 10 s,
at 120-s intervals. The voltage protocol is illustrated by the bottom traces. The corresponding whole-cell currents are
illustrated in the middle traces. pH
was
estimated simultaneously in the patched cell by measuring the
fluorescence ratio of BCECF and is shown by the top traces.
Data are representative of 3 similar
experiments.
Figure 6:
Relationship between the reversal
potential of tail currents (E) and the
transmembrane pH gradient. Osteoclasts were patched in the whole-cell
configuration using patch pipettes filled with NMG-Asp 6.0. The bath
was then perfused with NMG-Asp 7.5, NMG-Asp 6.8, or NMG-Asp 6.0. E
was determined using the following protocol:
the holding potential was -60 mV, outward current was first
activated by a 900-ms depolarizing pulse, chosen to activate the
current yet minimize pH
changes. Test pulses
lasting 350 ms were next applied between -90 mV and +30 mV
in +10-mV increments. A, representative tail currents
from an osteoclast with a capacitance of 60 pF patched with a 7-M
pipette. For clarity only currents at E
, one
inward and one outward current, have been illustrated. B,
curve fitting of tail currents like those illustrated in A was
used to determine the instantaneous tail current. Linear regression of
3-4 of these instantaneous currents on either side of the zero
current level was used to calculate E
, which is
plotted in panelBversus pH
. Each point represents the mean ±
S.E. of 3 experiments using cells from different animals. Where absent,
error bars were smaller than the symbol. The solid line is the
predicted H
equilibrium potential (E
) (see also Footnote
5).
Table 2also
demonstrates the effect of varying pH on the E
. Changes to the internal pH of osteoclasts
patched in the whole-cell mode were achieved at constant pH
by including a fixed concentration of
NH
/NH
in the pipette solution
and then altering the external NH
/NH
concentration to predetermined values. This protocol results in
rapid and reversible clamping of pH
, at levels which can be
predicted from the transmembrane NH
gradient (Table 2; see Grinstein et al.(1994) for
details). As was found for pH
, changes in pH
produced sizable shifts in the E
which
closely followed the predicted E
(see last
column of Table 2). Considered in aggregate, these findings
provide strong evidence that H
(equivalents) are the
species primarily responsible for the voltage-dependent outward
currents in acid-loaded cells.
Figure 7:
Effect
of pH on H
conductance.
Osteoclasts patched in the whole-cell configuration were clamped at a
holding potential of -60 mV. Pulses lasting 2.9 s were applied
between -90 and +75 mV in 15-mV increments at 30-s
intervals. The pipette contained solution NMG-Asp 6.0. The bath was
perfused with NMG-Asp 7.5 (opencircles), NMG-Asp 6.8 (solidcircles), or NMG-Asp 6.0 (open
triangles). Points were calculated by averaging the current during
the last 150 ms of each voltage pulse. To facilitate comparison between
experiments, the current generated at pH 7.5 during the +75-mV
pulse was set to 1 and all other currents are normalized to this value.
The relative currents of three cells from different animals are plotted versus the applied potential. Values are means ± S.E. (n = 3). Where absent, bars were smaller than the
symbol.
The
effect of decreased pH was also examined (Fig. 8).
Using the NH
/NH
gradient
protocol, changes to the internal pH were made in single cells at a
constant pH
= 7.5. Contrary to the effects of
altering pH
, internal acidification potentiated the
current, while shifting the activation threshold to more negative
potentials (Fig. 8). These findings can be partially explained
by the changes in driving force that accompany alteration in the
transmembrane pH gradient, lending further credence to the idea that
H
equivalents are the primary charge carriers. In
addition, however, pH
also exerts direct effects on the
permeability to H
. This property of the conductance
has been explored in more detail in macrophages (Kapus et al.,
1993).
Figure 8:
Effect of pH on
H
conductance. Osteoclasts patched in the whole-cell
configuration were clamped at a holding potential of -60 mV.
Pulses lasting 2.9 s were applied between -90 and +75 mV in
15-mV increments at 30-s intervals. The pipette contained solution
Cs-Asp/NH
-50. The bath was perfused with
Cs-Asp/NH
-1 (solid circles),
Cs-Asp/NH
-3 (open triangles),
Cs-Asp/NH
-9 (solid triangles), or
Cs-Asp/NH
-15 (open squares). The calculated
intracellular pH (see Grinstein et al.(1994) for details) is
indicated for each condition. Points were calculated by averaging the
current during the last 150 ms of each voltage pulse. Currents were
normalized as in Fig. 7. The relative currents of three cells
from different animals are plotted versus the applied
potential. Values are means ± S.E. (n = 3).
Where absent, bars were smaller than the symbol. The intracellular pH
was estimated as described under ``Experimental
Procedures.''
The ability to regulate pH within a tight
physiological range is crucial for normal cellular function. This is
particularly important for active cells such as osteoclasts, since the
products of their metabolism tend to deviate pH
away from
the normal range. The present studies analyzed some of the mechanisms
capable of acid extrusion from osteoclasts, which are likely involved
in pH
homeostasis during acidification of the osteoclast
microenvironment or activation of these cells by other means. As
previously reported (Teti et al., 1989; Hall and Chambers,
1990), osteoclasts cultured on glass coverslips had a functional,
amiloride-sensitive, Na
/H
antiporter
operating in the plasma membrane. However, unlike many other cells,
osteoclasts were also able to recover from an acute acid load in
Na
-free solutions, implicating the presence in these
cells of pH regulatory mechanisms other than the antiporter. Of note,
the Na
-independent recovery was observed mainly in
depolarized cells, suggesting that a voltage-sensitive, possibly
rheogenic process was involved.
Osteoclasts are known to be endowed
with plasmalemmal V-ATPases. These pumps operate physiologically to
translocate protons into the extracellular space and are necessary for
bone resorption. Our previous studies demonstrated that in peritoneal
macrophages, plasmalemmal V-ATPases play a major role in pH homeostasis (Swallow et al., 1990). Based on the common
lineage of these two cell types, it was anticipated that V-ATPases
would similarly contribute to pH
regulation in osteoclasts.
However, the recovery noted in Na
-free medium was
virtually resistant to inhibition by the specific V-ATPase inhibitor,
bafilomycin A
, under conditions and at concentrations where
the macrolide induced rapid dissipation of the pH gradient within the
intracellular organelles. Thus, the bafilomycin used was active and
achieved sufficiently high cellular levels to exert its inhibitory
effect. These data do not rule out the possibility that pump isoforms
may exist in the plasmalemma which are selectively resistant to
bafilomycin. However, bone resorption, which is ostensibly mediated by
V-ATPases at the ruffled membrane, has been shown to be susceptible to
bafilomycin A
(Sundquist et al., 1990).
Several
explanations may underlie our failure to detect V-ATPase-mediated
H extrusion from these cells. First, the cells were
cultured on glass coverslips rather than on bone slices (to permit
spectroscopic analysis) and this may preclude their exposure to the
normal physiological stimuli required for either physical or functional
expression of pumps on the plasmalemma. In other cells, environmental
stimuli are known to promote translocation of pumps present in
intracellular vesicles to the plasma membrane (Al-Awqati, 1986).
Alternatively, bone-dependent stimuli may induce association of
activator proteins (Xie et al., 1993) with the pump or
dissociate pump inhibitor molecules (Zhang et al., 1992).
Second, if the pumps are circumscribed to the portion of the membrane
in apposition to the substratum, they may be unable to contribute to
pH
regulation. Rapid acidification of the underlying
secretory space may kinetically or thermodynamically limit the activity
of such pumps, rendering them incapable of further H
extrusion upon cytosolic acidification. Cytochemical studies
defining the localization of V-ATPases under the conditions studied
should clarify the explanation. Further, osteoclasts cultured on glass
as in the present studies offer an excellent experimental model to
study H
-pump induction or up-regulation in seemingly
``quiescent'' osteoclasts.
The ability of the cells to
undergo Na-independent pH
recovery in
K
medium but not in NMG
suggested a
role for a voltage-dependent process, possibly a H
conductive pathway. Several lines of evidence support the notion
that H
conductance contributes to pH
recovery under these conditions. First, Zn
, an
inhibitor of conductive proton permeability in other systems,
reversibly prevents pH
recovery following an acid load.
Second, patch clamp studies using the whole-cell configuration showed
that an outwardly directed current could be induced when depolarizing
pulses were applied to osteoclasts. The current was reversibly blocked
by Zn
and was associated with cytosolic
alkalinization, the magnitude of which correlated with the size of the
current. Third, ion substitution experiments demonstrated that the
magnitude and direction of the current were virtually unaffected by
replacement of the major ionic species with large, impermeant organic
or inorganic ions. Importantly, Cs
was not inhibitory,
making it unlikely that a K
conductance was
responsible for the current (Ravesloot et al., 1989; Arkett et al., 1992). Further, the slow kinetics of the response were
distinctly different from those of the outward-rectifying K
current reported earlier in mammalian osteoclasts (Arkett et
al., 1992).
The magnitude of the current was found to depend on
the transmembrane pH gradient. Indeed, the E derived from analysis of the tail currents at varying pH
and/or pH
levels correlated well with the calculated E
, providing strong indication that
H
equivalents are the primary charge carriers. Thus,
when considered together, the results from both fluorescence
measurements and from electrophysiological studies provide convincing
evidence that pH
recovery in Na
-free
medium is mediated by a conductance that is extremely selective for
H
.
Is the magnitude of the current sufficient to
account for the observed changes in pH? Under conditions
that mimic those in the acid-loaded cells of Fig. 1Fig. 2Fig. 3, the ``steady state''
current at 75 mV averaged 373 ± 53 pA (n = 11),
equivalent to 6.7 pA/pF. At 0 mV, the approximate potential of cells in
K
media, the current density was about 5-fold lower (Fig. 4, Fig. 7, and Fig. 8). Assuming that the
current is carried entirely by H
equivalents and
considering the cellular volume, it is possible to predict the pH
change associated with the passage of current, if the buffering
capacity of the cells is known. The latter was estimated by pulsing
with ammonium and averaged 22.8 ± 3.5 mM/pH in
acid-loaded cells (pH
5.9-6.1) and 12.65 ± 2.8
mM/pH (n = 4) at physiological pH
.
Cellular volume was calculated from the measured average diameter of
the cells (
28 µm), assuming that adherent cells are
approximately hemispherical. When these parameters are taken into
consideration, the H
current recorded at
0 mV is
predicted to alkalinize the cells at a rate of 0.3 pH/min, which
compares favorably with the actual rates recorded fluorimetrically (Fig. 2B).
The exact physiological role for the
observed H conductance in osteoclasts has not been
established. A Zn
-sensitive H
conductance has recently been reported in peritoneal macrophages
(Kapus et al., 1993), in neutrophils (DeCoursey and Cherny,
1993), and in granulocytic HL60 cells (Demaurex et al., 1993).
The currents in these cell types share many similarities, including
outward rectification, sensitivity to internal and external pH, and
inhibition by heavy metals (DeCoursey and Cherny, 1993; Demaurex et
al., 1993; Kapus et al., 1993). One feature that is
common to all these cell types is their ability to undergo a
respiratory burst in response to stimulation. It has been suggested
that the conductance is required to extrude the protons that are
released by the oxidative pathway (Lukacs et al., 1993). Of
note, osteoclasts are similarly endowed with an NADPH oxidase and are
capable of a respiratory burst (Key et al, 1990; Garrett et al., 1990), so that the H
conductance may
serve a comparable function. While the conditions required for
stimulation of the conductance appear too extreme for it to activate in
the resting state, local acidification and/or a depolarization
generated by the purportedly electrogenic oxidase may suffice to gate
the current. Two stable levels of membrane potential have been reported
in osteoclasts, -70 and -15 mV (Mears, 1971; Ferrier et
al., 1986; Ravesloot et al., 1989; Sims and Dixon, 1989).
The depolarized state, believed to correspond to the activated cell,
would promote activation of the conductance at moderately acidic
pH
(i.e. 6.8-6.5, see Fig. 8), which
may result from metabolic acid generation. It is conceivable, therefore
that the voltage-sensitive H
conductance contributes
to translocation of acid equivalents during the early stages of
acidification of the lacunae. Subsequently, further extracellular
acidification would inactivate the conductance (Fig. 7),
preventing detrimental backflux of protons extruded by the pump.
In
summary, the present studies show that V-ATPases contribute minimally
to the recovery of cytoplasmic pH from intracellular acid loads.
Instead, the data are consistent with the presence of a
Zn- and membrane potential-sensitive
H
conductance in the plasma membrane of osteoclasts. In
this regard, it is noteworthy that there are reports in the literature
showing that Zn
can affect bone formation (Yamaguchi et al., 1987) and influences the course of osteoporosis
(Fushimi et al., 1993). The mechanism underlying this effect
of Zn
is unknown but could be related to inhibition
of the bone-resorbing activity of osteoclasts. In fact, a recent
preliminary report suggests that concentrations of Zn
expected to antagonize the conductance potently inhibit
osteoclastic bone resorption in vitro (Moonga and Dempster,
1994). Whether this is a direct effect on the H
conductance described herein is not known.