Bone as an ion exchange system: evidence for a pump-leak
mechanism devoted to the maintenance of high bone
K+
Alessandro
Rubinacci,
Fiorenza Dondi
Benelli,
Enrico
Borgo, and
Isabella
Villa
Bone Metabolic Unit, Scientific Institute H San Raffaele, Milano
20132, Italy
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ABSTRACT |
To provide evidence of
active accumulation of K+ in bone extracellular fluid
(BECF), electric currents driven by damaged living metatarsal bones of
weanling mice, immersed in physiological media at different
[K+], in the presence of blockers of the
K+ channels or of the Na+-K+-ATPase
inhibitor, were measured by means of a voltage-sensitive two-dimensional vibrating probe. At 4 mM extracellular K+
concentration ([K+]o), an
inward steady current density (7.85-38.53 µA/cm2)
was recorded at the damage site, which was significantly dependent on
[K+]o. At
[K+]o equal to that of BECF (25 mM), current density was reduced by 76%. At
[K+]o of 0 mM, the current density
showed an increase, which was hindered by tetraethylammonium (TEA).
Basal current density was reduced significantly after exposure to TEA
or BaCl2 and was unchanged after long- term exposure to
ouabain. By changing control medium with a chloride-free medium,
current density was reversed. The results support the view that
K+ excess in bone is maintained by a biologically active
cellular system. Because the osteocyte-bone lining cell syncytium was
at the origin of the current in bone, it is likely that this system controls the ionic composition of BECF.
ionic current; potassium; bone extracellular fluid
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INTRODUCTION |
BONE HAS AN EXTRACELLULAR FLUID (BECF) that flows
inside the osteocyte lacunae and the rich network of connecting
canalicula. BECF has a distinctly different ionic composition from
systemic extracellular fluid (ECF) and, in particular, a higher
concentration of potassium (K+; 25 mM vs. 4 mM,
respectively) (10, 25, 27, 35). The high content of K+ in
BECF was demonstrated many years ago (10, 16, 25, 27, 35), but neither
the mechanisms underlying K+ accumulation in bone nor the
physiological relevance of the K+ excess has been
explained. Bone K+ might participate in the maintenance of
acid-base equilibrium, being rapidly removed from the bone in response
to an acute acid load (7, 17). K+ concentrations might
control bone resorption (9) and matrix mineralization. In fact,
K+ reduces the incorporation of
CO2
3and alkali anions into
octacalcium phosphate, a transient intermediate in the
precipitation of the thermodynamically more stable hydroxyapatite (11), thus preventing the mineralization excess (31).
Passive physicochemical equilibrium (26) cannot explain the high
K+ levels. Because K+ is not bound to any
degree in the crystal hydration shells and does not interact
significantly with any of the solid phase constituents of formed bone
(10, 16), the elevated levels must be due to a metabolically active
partition (35). Several lines of evidence support this view. 1)
BECF K+ flows out of the bone surface after bone death (6).
2) Reduced medium pH depletes surface K+ of
cultured calvariae with respect to calcium (8). 3) The block of
the Na+- K+-ATPase alters the transport of
85Sr and 86Rb from BECF to ECF (22). 4)
The potential difference between BECF and ECF is consistent with an
energy-dependent accumulation of K+ within BECF (39, 40).
5) Finally, bone cells are polarized (1, 17, 38).
Despite the clear presence of a pronounced K+ gradient,
direct anatomic evidence for a barrier compartmentalizing the ionic composition of BECF from ECF is lacking. Recent observations, however,
point to the existence of a bone-lining cell-osteocyte syncitium (29)
that could perform such a function (17, 23, 30, 34). The presence of
gap junctions linking cells (29) and ion transport structures in cells
of osteoblast lineage (1, 20, 41) are in agreement with this view.
Classical electrophysiological methods have not been successfully
applied to the characterization of ion fluxes at the BECF-ECF interface. The hard structure of bone hinders the placement of microelectrodes within the endocanalicular space. However, the noninvasive, voltage-sensitive, vibrating probe system bypasses this
limitation because it measures that part of the ionic current loop
flowing outside the bone in the extracellular milieu (28). The probe,
therefore, can provide direct information on ion fluxes that occur
through bones immersed in physiological media. It has already been
observed that mechanically unloaded, living bone drives a steady ionic
current through a puncture site penetrating the diaphyseal cortex (4,
33, 34). Because the transcortical damage has the immediate effect of
exposing the BECF to ECF, ions are free to move down their
electrochemical gradients at the damaged site (leak), and the pumps,
devoted to the maintenance of the gradient, are activated. The
activation of a putative pump-leak system, first proposed by Borgens
(4), generates a detectable electric signal at the damage site (leak),
giving reliable information on the ionic exchanges occurring over the
intact bone surface. Because of its stability over time (4, 33, 34),
its temperature dependence (4), and its absence in dead bone (4, 33,
34) and after solubilization of the cell membrane (34), the injury current was ascribed to a biologically active cell system. A further study gave the first convincing evidence that the osteocyte-lining cell
system provides the driving force for the current (34). Because the
current is dependent on specific ion species, such as sodium, chloride
(4), and bicarbonate (33), which are known to display a concentration
gradient between BECF and ECF (25, 27, 32), a specific ion transport
mechanism at the BECF-ECF interface should be operative.
To explore the hypothesis that there is a partition system responsible
for K+ fluxes at the BECF-ECF interface, we measured the
electric current driven by living, mechanically unloaded, damaged bone.
The bone response to changes in the physiological media mimicking ECF
and containing different K+ concentrations and/or specific
K+ channels and Na+- K+-ATPase
blockers was tested.
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MATERIALS AND METHODS |
Incubation media.
The medium containing ECF K+ concentration
([K+]o = 4 mM) was defined as the
control medium, and media containing different K+
concentrations ([K+]o = 0 to 27 mM), different Cl
concentrations
([Cl
]o = 0 to 50 mM), and/or
drugs were defined as the test media (Tables
1-3).
Osmolarity was measured by an Osmostat Os 6020 pressure osmometer
(Damchi, Kyoto, Japan). Resistivity was measured by a HI 9033 multi-range conductivity meter (PBI International, Milano, Italy).
Reagents were purchased from Sigma (St. Louis, MO).
Bone samples.
Weanling mice (Swiss; Charles River, Italy) were killed with
CO2 in a gas chamber (Tecniplast, Varese, Italy). The back
limbs were amputated at the distal tibia epiphysis and immersed in an excision medium with the composition of the control medium (Table 1)
except that bicarbonate was substituted with
Na+-isethionate to assure the stability of pH during
surgical manipulations. The metatarsal bones were dissected intact from
the digit, with care taken to avoid damage to the bone surface. All
manipulations were carried out on samples immersed in the medium at
room temperature with an M3 surgical microscope (Wild, Zurich,
Switzerland). After the bone was freed of soft tissue ensheathments, a
50-µm-diameter hole, penetrating into the marrow cavity through the
diaphyseal cortex, was made with a thin stainless steel dental drill
(Mani, Matsutani Seisakusho, Ken, Japan). This involved a penetration to a depth of ~200 µm. The animal use was approved by the local Institutional Animal Care and Use Committee (Protocol no. TS 9501).
Experimental setup and data acquisition.
The experimental setup has been previously described (33, 34). Briefly,
all equipment (chamber, probe electrode, and microscope) was placed on
an M-TS 23 anti-vibration platform (Newport, Fountain Valley, CA). The
bone was held at the bottom of a specifically built Petri dish and
placed in an aluminum container over a Peltier heating chip kept at
37°C. The dish was filled with the appropriate prewarmed (37°C)
medium and was then covered by a light, white, mineral oil (Mineral
Oil, Sigma). Medium stability was monitored by a
pH-Po2-Pco2 automatic analyzer (Instrumentation
Laboratory, Lexington, MA) on aliquots taken at intervals during
readings. Temperature was monitored by a T801 thermoprobe (Radiometer,
Copenhagen, Denmark). A video camera (TK S200, JVC, Tokyo, Japan) was
connected through a BH Olympus microscope (Tokyo, Japan) to an RGB
monitor (EUM1491A, Mitsubishi, Tokyo, Japan). The area including the
vibrating electrode and the bone surface was viewed at an 18.23-mm
working distance using a D-achromat A4 × 4 lens (Olympus, Tokyo,
Japan). Light was provided through fiber-optic cables (Olympus)
connected to a light source (Intralux 5000, Volpi, Zurich, Switzerland).
The two-dimensional vibrating probe system has been described in detail
(15, 21, 28, 36, 37). Briefly, the reference electrode is a stationary
platinum black wire, and the probe is an insulated stainless steel
electrode (SS 300305A, Microprobe, Clarksburg, MD) with a platinum
black tip. This electroplated ball has a final diameter of ~3-5
µm (28). The probe is vibrated between two positions along the
x- and y-axes by means of a
-shaped linkage of three
rectangular piezoelectric bimorphs to which sinusoidal signals are
applied. The probe is vibrated at frequencies of 200-300 Hz for
the x-axis and 400-600 Hz for the y-axis over
distances twice the diameter of the platinum black ball. The
instrumentation measures the electrical potential differences between
the extremes of the probe excursion, along the directions of vibration,
with a lock-in amplifier. The signal is transferred via an
analog-digital board (DAS8, Keithley Metrabyte, Taunton, MA) to a
computer (PC CompaQ 3.86) for data acquisition, analysis, and storage.
The probe position is recorded, and software analyzes the two
orthogonal components of the measured signal, expressing them as an
average vector (Software lock-in amplifiers and vibrational assemblies were developed at the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, MA). The vectors are overlaid on a
digitized image of the preparation (PCVision Plus, Imaging Technology,
Bedford, MA). Each vector length represents the density of the current
at the measurement point, with directionality corresponding to the
arrowhead. By convention, the direction of the current flow is depicted
as normal to a net cation flux. If the current is carried by an anion,
the polarity must be interpreted as the reverse of that depicted by the
vector. The analog outputs of the system were also recorded on a
four-channel chart recorder (BD101, Kipp & Zonen, Delft, Holland).
To calibrate the probe, a 50 nA current was delivered into the control
medium through a glass micropipette (1 × 90 mm, GD-1, Narishige,
Tokyo, Japan) obtained with a micropipette puller (PB-7, Narishige) and
filled with 3 M KCl. The current source was placed (manipulator M-152,
Narishige) at two locations 150 µm away from the probe, in mutually
orthogonal directions. During experimentation when significant signals
out of phase with the probe vibration (quadrature output) developed,
the results were discarded because this usually indicated an artifact
caused, for example, by contact with the tissue.
Typically, vibrating probes were calibrated each day. The background
value was first measured by placing the probe far from the bone (>3
mm). Electrodes giving background values in excess of 0.5 µA/cm2 were rejected. The probe was moved using a 3D
micromanipulator (MO-203, Narishige) to the recording site,
~35-50 µm above the cortical hole, to map current densities.
The final current density was obtained by subtracting the background
value from the reading. The coefficient of variation, defined as
SD/average ×100 of the measurements obtained when the location
was kept constant, was 7%. Because the voltage probe technique is
based on a version of Ohm's Law (21), the current-dependent voltage
will be affected by the resistivity of the medium. The software
corrects for differences in resistivity with experimental treatment. It
assumes that the resistivity at the recording site is the same as the
bulk medium; given the lissajous movement of the probe, and the mixing
that results at a vibrational frequency of 200-400 Hz, this is a
valid assumption. Resistivity was measured at 37°C with a
conductivity meter (HI 9033 PBI International).
Experimental protocol.
After testing for the spatial distribution of current density over the
injury site, the probe was located at the point of maximal density.
This point was generally found over the center of the injury. Two
series of experiments were then performed. In the first series, the
current magnitude was first recorded in control medium (basal steady
current) and then in test media either with different K+
concentrations (Table1) and/or in the presence of blockers for K+ channels, [TEA (35mM, 50mM; Table 3) and
BaCl2 (2mM)] or in the presence of the
Na+-K+-ATPase inhibitor ouabain (1 mM). In the
second series, the current magnitude was first recorded in control
medium (basal steady current) and then in test media with different
Cl
concentrations (Table 2). The probe electrode was
positioned at the same location before and after media substitutions.
All experiments were performed at a controlled temperature (37°C),
pH (7.33 ± 0.06 at 37°C), and osmolarity (343 ± 11 mosM). Total
elapsed time for the K+ concentration experiments was ~1
h, whereas total elapsed time for the experiments with blockers was
~2 h.
Statistics.
Data were compared by a two-tailed Student's t-test for
paired observations. Data obtained from the experiments with different K+ concentrations were analyzed by means of ANOVA.
Differences were considered significant for P < 0.05.
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RESULTS |
An electric (ionic) current entering the site of damage was recorded in
all metatarsal bones tested (n = 98). Maximal current density
vector was normal to the bone surface, whereas moving along the bone,
current density decreased and current direction became progressively
parallel to the bone longitudinal axis (Fig. 1). The current appeared inward by the
convention we have discussed. In control medium, maximal current
density became steady after an initial slow decay. Control experiments
confirmed that the current remained stable for
2 h (Fig.
2, n = 4). At steady state, maximal
current density averaged 20.16 µA/cm2 and ranged from
7.85 to 38.53 µA/cm2 (n = 89; basal steady
current). When the control medium was exchanged with test media
containing different K+ concentrations, current density
changed. At [K+]o equal to (27 mM,
n = 2; 25 mM n = 8, P < 0.01; Fig
3, A and B) or lower than
(22 mM, n = 4, P < 0.01; 20 mM, n = 4, P < 0.01; Fig. 3, C and D) that of BECF (25 mM), the current density decreased significantly after an initial,
transient reversal. At 10 mM [K+]o,
current density decreased without an initial reversal (n = 4, P < 0.01; Fig. 3E), whereas at 0 mM
[K+]o, the current density
increased significantly (n = 7; P < 0.01; Fig.
3F) and remained steady for
70 min (data not shown). Current density returned to the basal steady values after exchange with control
medium. Current density changes on media exchange, expressed as a ratio
of current density measured in the test media at different K+ concentrations to that measured in control medium, were
significantly (Fisher's F = 32.14; P < 0.0001; Fig. 4) related to the medium K+ concentration. By applying Ohm's law on the current
density values for any given resistivity of the media at each different
K+ concentration, the calculated potential differences were
linearly related to the log
[K+]o (Fig.
5).

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Fig. 1.
Distribution pattern of steady current density at the damage site of
metatarsal bones of weanling mice immersed in control medium. Bone
damage is clearly visible as a round hole at the diaphyseal cortex.
Spatial resolving power of the two-dimensional, voltage-sensitive
vibrating probe allows detection of that part of the current loops
flowing outside of bone in the external milieu. Vectors represent
density (length), direction (angle), and sign (inward) of the net
current. By convention, direction of the current flow is that of a
cation flux. If the current is carried by an anion, the sign must be
interpreted as the reverse of that depicted by the vector. The
arrowhead corresponds to the point of measurement of the current (see
MATERIALS AND METHODS). Background value (<0.5
µA/cm2) is recognizable by the arrowhead far from the
bone surface. The scale of current density (10 µA/cm2) is
reported at top left.
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Fig. 2.
Values of ionic current density over time, entering (inward) the site
of damage of 4 mice metatarsal bones incubated in control medium (see
MATERIALS AND METHODS). Readings were obtained throughout
the experimental time that lasted ~2 h. Each symbol represents the
single measurement at that time for a single bone.
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Fig. 3.
Values of ionic current density over time, entering or leaving the site
of damage of mice metatarsal bones incubated in media containing
different K+concentrations (see MATERIALS AND
METHODS). When control medium containing 4 mM K+ was
changed with test media containing [K+] equal
to (27 mM, n = 2; 25 mM, n = 8; A and
B) or lower than (22 mM, n = 4; 20 mM, n = 4; C
and D) that of bone extracellular fluid (BECF: 25 mM), the
current density decreased after an initial transient reversal. At 10 mM
K+, current density decreased without an initial reversal
(n = 4: E), whereas at 0 mM K+, the current
density increased (n = 7; F). Changes of current
density were reversible, because basal injury current fully recovered
by returning to control medium. Each symbol represents the single
measurement at that time for a single bone.
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Fig. 4.
Relationship between injury current density and K+
concentrations in external milieu. Injury current density is expressed
as a ratio of plateau values recorded in test media at different
K+ concentrations to values recorded in control medium at
[K+] = 4 mM. Injury plateau current density was
significantly (Fisher's F = 32.14; P < 0.0001;
n = 27) dependent on [K+]o.
Data are expressed as means ( ) ± SD.
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Fig. 5.
Linear relationship (r2 = 0.79) between
potential differences ( V) recorded as soon as control medium was
substituted with test medium (see MATERIALS AND METHODS)
and K+ concentration (log
[K+]o). Data are expressed as means
( ) ± SD.
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Potassium channel blockers affected current density. When basal injury
current density was considered, the effect of K+ channels
blockers was inhibitory. In fact, the addition of 50 mM TEA, 35 mM TEA,
or 2 mM BaCl2 to the control medium significantly (P < 0.001) reduced basal steady current density by 60%
(n = 5), 40% (n = 3), and 30% (n = 4),
respectively. The reduction of the current density was reversible,
because basal steady current fully recovered after drug removal (Fig.
6, A-C). The inhibitory effect of
TEA was different at different
[K+]o. By increasing
[K+]o to 20 mM, current density was
reduced by 50% and was further reduced by 70% by the addition of 35 mM TEA (it did not matter when the drug was added) (Fig.
7, A and B). When TEA was present in both control and test media, no increased current density was observed by removing K+ from the external medium, whereas
when the TEA addition was coupled to the test medium with
[K+]o = 0 mM, current density was
reduced by 58% (Fig. 7, A and B).

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Fig. 6.
Effect of 50 mM tetraethylammonium chloride (TEA, n = 5;
A), 35 mM TEA (n = 3; B), and 2 mM
BaCl2 (n = 4; C) on injury current density
in mouse metatarsal bone. The reduction of current density was
reversible for TEA and BaCl2, because basal steady current
fully recovered after drug removal. In the experiments with ouabain,
basal steady current did not recover after drug removal. Each symbol
represents the single measurement at that time for a single bone.
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Fig. 7.
Effects of 35 mM TEA on injury current density in mouse
metatarsal bone with different K+concentrations in
the external milieu. A: dotted bars represent percent reduction
of injury current density when TEA was added either to control
medium (4 mM K+) or to test media containing 0 mM or 20 mM
K+. Open bars represent percent changes of injury current
density when [K+]o was changed from
4 mM (control medium) to 0 mM or 20 mM (test media) without TEA. When
TEA was added to test media at
[K+]o = 0 mM, no increased current
density was observed. B: hatched bars represent percent changes
of injury current density when TEA was already present before change to
test media containing 0 mM K+ or 20 mM K+ (see
MATERIALS AND METHODS). Because TEA was already present in
control medium, no further reduction of basal injury current was
observed at [K+]o = 0 mM. Each
symbol represents the single measurement at that time for a single
bone. Data are expressed as means ± SD. * P < 0.05; **
P < 0.01.
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When the bones were exposed to 1 mM ouabain for short times (~30
min), there was an apparent decrease of current density (Fig. 8A, n = 4) that was not
confirmed by extending the exposition time to over 90 min (Fig.
8B, n = 3). In fact, after a long exposition time that
was chosen to assure the complete permeation of the Na+-K+-ATPase inhibitor into the bone, current
density remained stable for
2 h (Fig. 8B), as it did under
control conditions (Fig. 2).

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Fig. 8.
Injury current densities in mouse metatarsal bone after brief
(n = 4; A) and long (n = 3; B) exposure
to 1 mM ouabain. Under long-term exposure, the medium containing
ouabain was changed with a fresh one to assure the stability of its
physicochemical characteristics. Each symbol represents the single
measurement at that time for a single bone.
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When the control medium was changed with test media containing
different Cl
concentrations, current density changed.
When [Cl
]o was lower than
ECF (100 mM), the current density decreased in a
nonconsistent manner. At
[Cl
]o equal to 50 mM
(n = 5) or lower (25 mM, n = 6; 10 mM, n = 6; 4 mM, n = 7), current decreased after a transient reversal that did not occur in all bones tested (Fig. 9,
A-D). At 0 mM
[Cl
]o, current density
reversed in all bones (n = 5) (Fig. 9E). By reconstituting the control conditions
([Cl
]o =100 mM), current
density returned to the basal steady values after a transient increase.
Current density changes on media exchange, expressed as the ratio of
current density measured in the test media at different
Cl
concentrations to that measured in control, were
not significantly related to the medium Cl
concentration (data not shown).

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Fig. 9.
Values of ionic current density over time, entering or leaving the site
of damage of mice metatarsal bones incubated in media containing
different Cl concentrations (see MATERIALS AND
METHODS). When control medium containing 100 mM
Cl was changed with test media containing a
[Cl ] lower (50 mM, n = 5; 25 mM,
n = 6 ; 10 mM, n = 4; 4 mM, n = 7; A-D) than that of BECF (130 mM), current density
decreased but not consistently. At 0 mM
[Cl ]o, current density
reversed in all tested bones (n = 5; E). Changes of
current density were reversible, because basal injury current fully
recovered at return to control medium. Each symbol represents the
single measurement at that time for a single bone.
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DISCUSSION |
This study confirmed previous observations (4, 33, 34) that damaged
bone generates a steady electric (ionic) inward current at the damage
site. According to the general interpretative model of electric
currents at an injury site mapped with a vibrating probe (3), the
damage of the cortex creates a "point sink" of the current,
partially shorting out the potential difference between BECF and ECF at
the site of damage. Thus ions are free to move along their
electrochemical gradients through this low-resistance pathway. As a
consequence, a driving force is activated to maintain the ionic
composition of the BECF. The activation of this system generates the
detectable electric current at the damage site with an inward
direction. The injury current is sustained over time by a driving force
provided by a cellular battery (4, 33, 34). The cell lineage that
accomplishes the task of maintaining the current over time is the
osteocyte-lining cell system (34) that appears to compartmentalize BECF
from ECF (6, 17, 23, 24). At the intact bone surfaces, the fluxes of
anions and cations exchanged between BECF and ECF are electrically
neutral, and no current densities are detectable (4, 33, 34).
The critical components of the injury current are the ionic species for
which a concentration gradient exists between the two extracellular
ionic compartments, i.e., BECF and ECF (25). Ionic substitution
experiments demonstrated in fact that chloride (4), sodium (4, 34), and
bicarbonate (33) are carriers of the current. This study made
progress in determining the ionic species carrying the
current, and it was specifically addressed to K+ , for
which the different composition between BECF and ECF has been a puzzle
for many years (10, 25-27). By demonstrating that injury current
varied by modifying the [K+] gradient between
the BECF and ECF, this study gives convincing proof that an ion
transport system is operative in bone to control the K+
content of bone microenvironment. By also demonstrating that injury
steady current was reduced after the bone exposure to specific K+channel blockers, this study suggests that K+
channels should be involved in fine-tuning bone K+ balance.
The model.
At the present state of knowledge, an interpretative model of ion
fluxes between bone and plasma that could describe current density
changes as a function of K+ concentration in the external
medium could be only speculative. It is based on the following
observations:
a potential difference exists between the ECF and BECF; this
potential difference is negative with respect to ECF (39); and the
polarity of the potential difference is consistent with an active
transport system for K+ into BECF (39), as postulated by
Neuman (25, 27);
bone surface is electronegatively charged (12, 13);
the osteoblast appears to behave like an epithelial cell, with a
Na+-K+-ATPase and
Na+/H+exchanger at the basolateral membrane
facing the systemic circulation, and
Cl
/HCO
3 and
Cl
and K+ channels facing the mineral
side; by allowing K+ and Cl
exit through
the apical membrane to the BECF (17), these exchangers determine the
alkalinization of the bone microenvironment, thus favoring the process
of mineralization that is dependent on alkaline pH;
although the cells of the osteogenic lineage constitute a
functional syncytium that extends up to the vascular endothelium (29),
they present gaps large enough to allow the passage of macromolecules
(horseradish peroxidase, antimony compounds, and lanthanum nitrate)
(19, 24); in particular, the lining cells that cover endosteal,
periosteal, and haversian surfaces and that are considered to function
as a selective barrier between BECF and ECF (17, 23) by sealing up the
endocanalicular space, display paracellular spaces and could even
retract in the presence of parathyroid hormone. Ions could therefore
leave BECF through a paracellular pathway along a concentration gradient.
The model, as shown in Fig. 10,
represents the cell of the osteogenic lineage, the distribution of the
channels and transporters, and the source and direction of the measured
extracellular net current. Intracellular currents were not considered
in the model because they are beyond the scope of the present study.

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Fig. 10.
The model of bone extracellular fluid (BECF) and extracellular fluid
(ECF) represents cells of osteogenic lineage, distribution of channels
and transporters, and source and direction of measured extracellular
net current. Intracellular currents were not considered in the model
because they were beyond the scope of the present study (see
DISCUSSION for extensive explanation).
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According to the general distribution of electric currents at an injury
site mapped with a vibrating probe (3), the model assumes that the
damage tends to short out the potential difference at the damage site
(the point sink of the current) and that the K+ current loop originates from the intact portions of bone
where the K+ concentration gradient drives K+
out from BECF through the paracellular spaces. The return pathway of
the current loop into the bone occurs at the point sink where the
electrical potential difference (39) between BECF and ECF has been
shorted out by the damage. Because of the potential difference of BECF
(negative) with respect to ECF, K+ is driven back into
BECF, thus closing the K+ current loop. As far as the
osteocyte-lining cell system is viable, the loop can be maintained, and
the relative current can be measured (4, 33, 34). Current density was
in fact increased by removing K+ from the bath, and it was
reduced by placing the ECF K+ concentration equal or
proximal to the BECF one. By removing K+ from the bath, the
outward driving force for K+ exit at the intact portions of
bone is amplified (origin of the current loop) with subsequent
enhancement of the cation return pathway and associated current at the
point sink. By placing external K+ concentration equal to
the BECF one, the outward driving force for K+ exit at the
intact portions of bone is reduced, with subsequent decrease of the
return pathway and associated current at the point sink. Because
current density was also reduced by blockers of the K+
channels such as BaCl2 and TEA, K+ should leave
the osteocyte-lining cell system and accumulate into the
endocanalicular space and lacunae. Because of the polarity of the cells
from the osteogenic lineage, the block of K+ channels
facing the mineral side (BECF) prevents the transcellular exchange of
K+ between BECF and ECF and the associated return pathway
of the current at the point sink. When TEA was already present in
control medium, the model assumes that K+ concentration in
BECF is reduced, as well as the BECF-ECF concentration gradient for
K+. K+ could in fact only leak from bone both
at the damage site and through the paracellular spaces, as it is not
transported back into the BECF by the cells because of the block of the
K+ channels. By blocking the channels, it is likely that
K+ accumulates in the cell, thus blocking the still
undefined active transporters. Under this nonphysiological condition,
BECF content of K+ would change, acquiring the same
concentration as that of the infinite bath (ECF), because the bone
tissue is unable to refill its subdiaphyseal cortex reservoir of
K+. The reduction of the K+ content into the
bone under TEA exposure could therefore explain why no increased
current density was observed when K+ was removed from the
bath. Of course, when TEA was already present in control medium, no
further reduction of the basal injury current could be observed.
This study did not succeed in demonstrating any involvement of the
ouabain-sensitive Na+-K+-ATPase. The small
decay in current density observed after brief exposure could be
aspecific, as it was not confirmed by the long exposure to the
inhibitor. It is therefore likely that the
Na+-K+-ATPase pump is not part of the
homeostatic mechanism responsible for the generation of the ionic
gradient between BECF and ECF, as early ouabain experiments (35) as
well as the more recent ones demonstrated (40).
The proposed model is confirmed by Cl
substitution
experiments. According to the model just reported, the reversal of the injury current in Cl
free medium is due to
magnification of the Cl
concentration gradient
between BECF and ECF that drives Cl
out from BECF
through the paracellular spaces at the intact surfaces. The reversal of
the polarity of BECF with respect to ECF could drive
Cl
back into the endocanalicular fluid compartment
at the point sink, thus closing the Cl
current loop
(return pathway). The prevailing contribution of the
Cl
current loop leads to the observed net current
reversal at the point sink, because the direction of current flow is
defined, by convention, as the direction in which positive ions move
(see Experimental set-up and data acquisition). Because current
density was not linearly related to Cl
concentration, it is likely that several transporters that are devoted
to the maintenance of the physiological ionic environment of the
endocanalicular space are activated.
The meaning of the current in bone.
The ionic current described in this study could be involved in the
site-directed bone remodeling and repair processes. In fact, it can be
speculated that a point sink of current could be induced by the
osteoclast resorption of the bone matrix as well as by the damage of
the bone cortex. In intact bone, it can be hypothesized that osteoclast
activity induces a microinjury current at the resorption lacuna
by exposing BECF to ECF. The current would be sustained by the
osteocyte-lining cell system of the surrounding bone until the opening
of the endocanalicular space to the plasma ceased to be sealed by
the subsequent osteoblast matrix deposition. Under the influence of
specific ionic gradients associated with the current, a directed
migration of the osteoblasts might occur. In fact, osteoblasts exhibit
cathodal galvanotaxis in vitro by migrating along the axis of an
electrical field produced by an exogenously applied constant current of
the same order of magnitude as that endogenously generated (14).
Moreover, by exposing BECF to ECF in the resorption lacunae, osteoclast
activity would locally reduce the K+ content. It is
therefore likely that the drop of external K+ would cause
an increment of the membrane potential difference of the
osteocyte-lining cell system that would activate Cl
channels and induce a consequent Cl
efflux from the
cell cytoplasm. Cl
exit should in turn
increase HCO
3 in the lacunae and
stimulate mineralization. In damaged bone, the phenomena
described above are most likely reproduced in a larger scale.
Conclusion.
This study confirms that a complex ion transport system is operative in
bone to control the ionic composition of the bone microenvironment.
This study provides evidence that K+ is accumulated
in BECF and that K+ channels are involved in fine-tuning
bone K+ balance. Although a working model of
transcellular ionic movement in bone is, as yet, incomplete and
speculative, this study strengthens the view that bone is an
active ion-exchanging system that participates in mineral homeostasis
and acid-base equilibrium maintenance (2). The endogenous
generation of an ionic current in bone as soon as BECF is exposed to
ECF by both osteoclast activity and damage could be part of the
mechanisms of site-directed bone remodeling and repair, respectively.
 |
ACKNOWLEDGEMENTS |
We thank Peter J. S. Smith, Biocurrent Research Center, Marine
Biological Laboratory (Woods Hole, MA) for criticism and editorial comments. We are greatly indebted to Gastone Marotti, Department of
Anatomy, University of Modena, and to Antonio Zaza, Department of
Physiology, University of Milano, for helpful discussion and scientific
support in the development of the interpretative model of ion fluxes in bone.
 |
FOOTNOTES |
This work was supported in part (40%) by the Italian Ministry of
University and Scientific Research MURST.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Rubinacci, Bone Metabolic Unit, Scientific Institute H, San
Raffaele,Via Olgettina 60, 20132 Milano, Italy (E-mail:
alessandro.rubinacci{at}hsr.it).
Received 15 July 1999; accepted in final form 10 September 1999.
 |
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