Department of Physiology and Division of Oral Biology, School of Dentistry, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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Insulin-like growth factor I (IGF-I) is thought to stimulate bone resorption indirectly through a primary effect on osteoblasts, which in turn activate osteoclasts by as-yet-unidentified mechanisms. Small decreases in extracellular pH (pHo) dramatically increase the resorptive activity of osteoclasts. Our purpose was to characterize the effect of IGF-I on acid production by osteoblastic cells. When confluent, UMR-106 osteoblast-like cells and rat calvarial cells acidified the compartment beneath them. Superfusion with IGF-I caused a further decrease in pHo. To investigate the mechanism, we monitored acid efflux from subconfluent cultures. IGF-I rapidly increased net efflux of H+ equivalents in a concentration-dependent manner. IGF-II (10 nM) evoked a smaller response than IGF-I (10 nM). The response to IGF-I was partially dependent on extracellular Na+, but not glucose, and exhibited little if any desensitization. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, abolished the response to IGF-I but not to parathyroid hormone. Thus IGF-I enhances acid efflux from osteoblastic cells, via a signaling pathway dependent on activation of phosphatidylinositol 3-kinase. In vivo, acidification of the compartment between the osteogenic cell layer and the bone matrix may affect diverse processes, including mineralization and osteoclastic bone resorption.
extracellular pH; osteoblasts; microphysiometry; phosphatidylinositol 3-kinase; wortmannin
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INTRODUCTION |
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REMODELING OF THE SKELETON is a complex process regulated by interactions among bone cells, extracellular matrix, and soluble factors. Insulin-like growth factors (IGFs) IGF-I and IGF-II are among the most abundant growth factors present in the bone matrix and play important roles in skeletal growth and development. IGF-I stimulates the proliferation of preosteoblastic cells, the synthesis of collagen by mature osteoblasts, and the formation of bone in vivo (8, 9).
In contrast to the well-characterized anabolic actions of IGF-I, its effects on bone resorption are less well understood. Parenteral administration of IGF-I to human subjects rapidly increases serum and urinary markers of bone resorption and formation (12, 16). In addition, IGF-I stimulates osteoclast formation in organ cultures of mouse metacarpals/metatarsals (27) and in cultures of mouse hematopoietic blast cells (18). Furthermore, IGF-I stimulates resorption by mixed populations of mouse or rabbit bone cells cultured on dentin slices (14, 18). Similarly, when rodent bone cells were cultured on ivory slices for 24 h, IGFs increased both the number and volume of osteoclastic resorption lacunae (15). Interestingly, IGF-I has no effect on the resorptive activity of isolated rodent osteoclasts, but responsiveness to IGFs is restored when osteoclasts are cocultured with osteoblastic cells. Thus IGFs appear to stimulate the resorptive activity of osteoclasts indirectly via a primary effect on other cell types such as osteoblasts (15).
Resorptive agents (such as parathyroid hormone) act on osteoblasts, which in turn stimulate osteoclast formation and activity. This process may involve enhanced osteoblastic expression of cell-surface signaling molecules or release of soluble mediators. In this regard, decreasing extracellular pH (pHo) markedly enhances osteoclastic resorption (1, 7, 26). In vitro, even slight acidification of the culture medium can dramatically increase the number of resorption pits formed by osteoclasts (2). Thus osteoblast-mediated acidification of their environment may rapidly increase the resorptive activity of mature osteoclasts (1).
The purpose of our study was to determine the effects of IGF-I on acid efflux from osteoblastic cells by use of a Cytosensor microphysiometer to monitor real time changes in pHo beneath osteoblastic cells. We examined the ability of osteoblastic cells to acidify an extracellular compartment, characterized the effects of IGF-I on this process, and investigated the underlying signaling pathways.
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METHODS |
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Materials and solutions.
-Minimum essential medium (
-MEM, cat. no. 12571) buffered with
HCO
3 (26 mM), phosphate-buffered
saline (cat. no. 14040), heat-inactivated fetal bovine serum (FBS, cat. no. 26140), antibiotic solution (penicillin 10,000 units/ml;
streptomycin 10,000 µg/ml; and amphotericin B 25 µg/ml, cat. no.
15240), trypsin solution [nominally
Ca2+- and
Mg2+-free, 0.05% trypsin, and
0.53 mM EDTA (cat. no. 25300)], Dulbecco's modified Eagle's
medium (DMEM, cat. no. 23800), and
HCO
3-free MEM (used in standard
superfusion medium, cat. no. 41500) were obtained from Gibco
Laboratories (Burlington, ON, Canada). Bovine albumin (fraction V,
fatty acid free) was from Boehringer Mannheim (Laval, QC, Canada; cat.
no. 775835). Collagenase type II (cat. no. C-6885), 2-deoxyglucose,
N-methyl-D-glucamine
(NMG), and cytochalasin B were obtained from Sigma (St. Louis, MO).
IGF-I and -II (recombinant human, cat. nos. H-3102 and H-7020,
respectively) and parathyroid hormone (PTH) fragment [human,
PTH-(1
34), cat. no. H-4835] were obtained from Bachem
(Torrance, CA). Stock solutions of IGF-I and -II were prepared in
standard superfusion medium (see next paragraph) and
stored in aliquots in
20°C. For experiments investigating the dependence of proton efflux on extracellular glucose or
Na+, a stock solution of IGF-I was
prepared in Na+- and glucose-free
buffer and stored in aliquots at
80°C. A stock solution of
PTH-(1
34) was prepared in 0.005 N acetic acid with 1 mg/ml bovine
albumin and stored in aliquots at
80°C. Phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin, and LY-294002
[2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] were
obtained from Calbiochem (La Jolla, CA). Stock solutions of wortmannin
and LY-294002 were prepared in dimethyl sulfoxide and stored in
aliquots at
80°C.
2-Deoxy-D-[1,2-N-3H]glucose
(26 Ci/mmol) was purchased from Du Pont Canada (Lachine, QC).
Cells and culture.
The clonal osteoblast-like cell line UMR-106, originally isolated from
a rat osteosarcoma (20), was obtained from the American Type Culture
Collection (Rockville, MD). These cells exhibit properties of
osteoblasts, including type I collagen production, high alkaline phosphatase activity, responsiveness to PTH and IGF-I, and formation of
mineralized tumors in rats (20, 22). UMR-106 cells were subcultured
twice weekly in -MEM supplemented with FBS (10% vol/vol) and
antibiotics (1% vol/vol).
Measurement of pHo and acidification
rate.
Cells adhering to the polycarbonate membranes were placed in microflow
chambers and positioned above silicon-based potentiometric sensors,
which can detect changes in pHo of
as little as 103 units
(Cytosensor microphysiometer, Molecular Devices, Sunnyvale, CA) (17).
Cells were continuously superfused at a rate of 100 µl/min with the
indicated solution at 37°C. Superfusion solutions with low
buffering power were used to enhance the changes in
pHo resulting from small
alterations in efflux of H+ from
cells. Each of the four microflow chambers was supplied with
superfusion solution from one of two reservoirs. Flow was regulated by
a computer-controlled valve, and the lag between a valve switch and the
arrival of solution at the microflow chamber was 4-5 s. The
surface potential of each silicon sensor, corresponding to the
pHo, was plotted initially as a
voltage-time trace. At 37°C, a change of 61 mV corresponds to a
change in pHo of 1 unit. During
experiments in which the effects of IGF-I on
pHo were monitored, cells were
superfused continuously with standard medium or IGF-I (in standard medium).
Scanning electron and light microscopy. The preparation of specimens for scanning electron microscopy was based on techniques of Brunk et al. (6). Following selected experiments, membranes with adherent cells were removed from the microflow chambers and placed in fixative (3% glutaraldehyde in 0.1 M cacodylate buffer) at room temperature for 1 h. The membranes were then rinsed in cacodylate buffer and postfixed (1% OsO4 in cacodylate buffer) for 1 h. Membranes were subsequently rinsed in water and dehydrated through a graded series of ethanol. Specimens were then critical point-dried and coated with gold-palladium (8 nm) in a vacuum. Membranes were examined using a Hitachi 35 scanning electron microscope operated at 20 kV. In some other experiments, cultures were fixed in ethanol, embedded in paraffin, sectioned perpendicular to the membrane, stained with hematoxylin and eosin, and examined by light microscopy.
Measurement of glucose uptake.
Initial rates of glucose uptake by UMR-106 cells were measured using
radiolabeled 2-deoxyglucose, as described previously (22). Cultures
were treated with IGF-I (10 nM) or vehicle in conditioned serum-free
-MEM for 15 min at 37°C in 5%
CO2-95% air. Cultures were then
washed and incubated for 1 min at 23°C with
2-deoxy-D-[3H]glucose
(60 µM, specific activity adjusted with unlabeled 2-deoxyglucose to
3.3 mCi/mmol) in transport buffer. Transport buffer consisted of (in
mM) 134 NaCl, 5.4 KCl, 1.8 CaCl2,
0.8 MgSO4, and 20 HEPES, adjusted
to pH 7.30 ± 0.02 and 290 ± 5 mosM/l. Where indicated, cytochalasin
B (10 µM) was included in the transport buffer to inhibit facilitated
hexose transport. An aliquot of incubation buffer was collected at the
end of each uptake incubation. Incubations were terminated by washing
cultures with ice-cold isosmotic Tris-sucrose solution. Cells were
harvested by osmotic lysis (1 ml water/dish) and mechanical scraping.
An aliquot (100 µl) of the cell harvest was used for protein
measurement, and the remainder was combined with scintillation
cocktail. The radioactive contents of the buffer and cells were
measured using liquid scintillation counting.
Statistics. Acidification rates were normalized as a percentage of basal rates in standard superfusion medium before addition of test substance or change of superfusion solution. This normalization compensated for differences in cell numbers among the four chambers. Results are presented as representative traces or as means ± SE. Comparisons between two means were performed using the Student's t-test. Comparisons among three or more means were performed by ANOVA followed by a Tukey-Kramer test for multiple comparisons. Differences were accepted as statistically significant at P < 0.05.
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RESULTS |
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Effects of IGF-I on pHo beneath UMR-106
osteoblast-like cells.
UMR-106 cells were cultured at high or low density on porous
polycarbonate membranes. Cultures were placed in microflow chambers, and pHo was monitored using
sensors located beneath the membranes. In this set of experiments,
cultures were continuously superfused with standard medium. IGF-I (10 nM in standard medium) induced a rapid decrease in
pHo beneath high-density cultures
(the maximum decrease was 0.07 ± 0.01 pH units below basal,
n = 19) (Fig.
1A). This drop in pHo was sustained for
the remainder of the 21-min exposure to IGF-I. Upon washout of IGF-I,
pHo slowly recovered, returning to
basal levels within ~1 h. In contrast, superfusion of low-density
cultures with IGF-I, or high-density cultures with vehicle, did not
cause any significant change in
pHo (as summarized in Fig.
1B).
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Effect of IGF-I on proton efflux from UMR-106 osteoblast-like cells.
Next, we considered the possibility that the effects of IGF-I on
pHo were due to increased efflux
of H+ equivalents after
stimulation of cells by growth factor. For these and all subsequent
studies, acidification rate was determined from the rate of decrease in
pHo when superfusion of the
cultures was periodically interrupted, and this value was used to
calculate proton efflux. Superfusion of cells with IGF-I (10 nM) caused a rapid increase in proton efflux from both high- and low-density cultures. However, changes in proton efflux caused by the associated drop in pHo made data recorded
from high-density cultures difficult to
interpret2.
Therefore, low-density cultures were used for the detailed studies of
proton efflux presented below. The increase in proton efflux from
low-density cultures was sustained in the presence of IGF-I (Fig.
4A).
IGF-I (10 nM) induced a maximum increase in proton efflux of 16 ± 1% above basal levels (n = 27). In contrast, superfusion of cells with vehicle did not change
proton efflux (Fig. 4A). In standard
superfusion medium, basal acidification rates were 0.076 ± 0.004 pH
units/min (n = 111), which
correspond to a proton efflux per cell sample of 0.30 ± 0.02 nmol/min.
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Evidence for involvement of the IGF-I receptor.
To investigate the pathway underlying the effect of IGF-I on proton
efflux from UMR-106 cells, we first examined its dependence on IGF-I
concentration. An increase in proton efflux was observed at IGF-I
concentrations 10 pM. The concentration of IGF-I required to elicit a
half-maximal response (EC50) was
~300 pM, with maximum increase in proton efflux occurring at
10-30 nM IGF-I (Fig. 5). The concentration dependence of this
response is in keeping with the values of
EC50 reported for responses
mediated by the IGF-I receptor in other systems (29).
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Dependence of IGF-I-induced acidification response on extracellular
Na+.
We next investigated the mechanisms underlying the IGF-I-induced
increase in proton efflux from UMR-106 cells.
Na+/H+
exchange, mediated by NHE-1, is an important pathway for proton efflux
in osteoblasts (13). To investigate the involvement of Na+-dependent processes such as
NHE-1, we compared the response of cells to IGF-I (10 nM) in the
presence and absence of extracellular Na+. Cells were initially
superfused with standard medium. When cells were then superfused with
control (Na+-containing) buffer,
IGF-I induced a maximum increase in proton efflux of 27 ± 4% above
basal (n = 5; Table
1). When parallel cell samples were
superfused with nominally Na+-free
buffer, the basal proton efflux dropped to 91 ± 8% of control levels, and IGF-I induced an increase in proton efflux of 15 ± 4%
above basal (significantly less than the response to IGF-I in
Na+-containing buffer). When these
cells were once again superfused with standard medium (which contains
physiological
[Na+]o),
there was a transient overshoot in proton efflux before it returned to
the level seen in control cells (i.e., cells exposed to IGF-I in
Na+-containing buffer). In
summary, removal of Na+ had little
effect on basal proton efflux but significantly suppressed the increase
in acid efflux induced by IGF-I.
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Role of glucose in IGF-I-induced acidification response. We considered the possibility that the IGF-I-induced increase in proton efflux was due to enhanced metabolism of glucose, perhaps secondary to an increase in glucose uptake. Thus we compared the acidification response of cells to IGF-I (10 nM) in the presence and absence of extracellular glucose. UMR-106 cells were initially superfused with standard medium. When cells were then superfused with control (glucose-containing) medium, IGF-I induced a maximum increase in proton efflux of 13 ± 2% above basal (n = 3; Table 1). When parallel cell samples were superfused with glucose-free medium, there was a large and rapid decrease in basal proton efflux to 23 ± 6% of control levels. This finding indicates that basal metabolic acid production by these cells is strongly dependent on glucose. However, in the absence of glucose, IGF-I still induced a marked increase in proton efflux of 12 ± 1% above basal (not significantly different from the response to IGF-I in control glucose-containing medium). When the cells were once again superfused with glucose-containing standard medium, proton efflux returned slowly to basal levels. In summary, although removal of extracellular glucose caused a large, reversible decrease in basal proton efflux, IGF-I still induced a substantial increase in proton efflux.
IGF-I, like insulin, increases facilitated hexose transport activity in a number of cell types, including osteoblasts (22, 25). Therefore, we considered the possibility that the IGF-I-induced increase in proton efflux was due to an increase in the rate of glucose uptake. To investigate the effect of IGF-I on glucose uptake, we measured the initial rate of uptake of 2-deoxyglucose. UMR-106 cells were pretreated with IGF-I (10 nM) or vehicle for 15 min. Uptake was then assessed using 1-min incubations with 2-deoxy-[3H]glucose in the presence or absence of the facilitated hexose transport inhibitor cytochalasin B (10 µM). As expected, cytochalasin B blocked glucose uptake. In contrast, IGF-I had no significant effect on the initial rate of 2-deoxyglucose uptake (Fig. 7). Taken together, these findings indicate that the IGF-I-induced increase in proton efflux does not arise simply from an increase in the rate of glucose uptake.
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Role of PI 3-kinase in the IGF-I-induced acidification response.
In other systems, PI 3-kinase mediates many of the metabolic responses
to IGF-I (25). To assess the involvement of PI 3-kinase in the
IGF-I-induced acidification response, we used wortmannin, a potent
inhibitor of PI 3-kinase (10). UMR-106 cells were initially treated
with wortmannin (100 nM) or vehicle in standard superfusion medium for
10 min. Wortmannin by itself did not change basal proton efflux. Cells
were then treated with IGF-I (10 nM) for 20-21 min in the presence
of wortmannin or vehicle. Under these conditions, wortmannin virtually
abolished the IGF-I-induced increase in proton efflux from a maximum of
15 ± 3% in vehicle-treated control cells to 0.9 ± 0.6%
in wortmannin-treated cells (n = 4, Fig. 8). No change in proton efflux was
observed upon washout of wortmannin.
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Effect of IGF-I on pHo and proton efflux
from osteoblasts isolated from rat calvaria.
Responses to IGF-I were also examined using first-passage
osteoblast-enriched cultures obtained from rat calvaria. When
high-density calvarial cell cultures were continuously superfused with
standard superfusion medium, IGF-I (10 nM) rapidly induced a sustained decrease in pHo beneath the cell
layer (the maximum decrease was 0.025 ± 0.003 pH units below basal,
n = 7) (Fig.
9A).
Superfusion of high-density cultures with vehicle did not cause any
significant change in pHo. Thus,
as in UMR-106 cells, IGF-I induced a sustained decrease in
pHo beneath high-density cultures
of calvarial cells.
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DISCUSSION |
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Confluent osteoblastic cells acidify the compartment beneath the cell layer. In the present study, we used a Cytosensor microphysiometer to monitor changes in pHo beneath cultures of UMR-106 osteoblast-like and rat calvarial cells. The compartment between confluent osteoblastic cell layers and the silicon sensor was acidified under basal conditions, and the pHo of this compartment was further decreased in response to IGF-I. Decreases in pHo were observed beneath confluent high-density cell layers but not beneath subconfluent low-density cell layers. This finding suggests that confluent layers act as barriers to diffusion, resulting in accumulation of protons beneath the cell layer. This model is in keeping with early suggestions that the "internal" pH of bone must be considerably lower than 7.4 (19).
The decrease in pHo induced by IGF-I may be due to an increase in net efflux of H+ equivalents. Alternatively, IGF-I may decrease the paracellular passage of H+ equivalents across the cell layer. However, IGF-I caused increases in proton efflux, which exhibited several similarities to the effect of IGF-I on pHo. These include a similar pattern, time course, and lack of desensitization, suggesting that the effect of IGF-I on pHo is due to an increase in proton efflux from the cells. During exposure to IGF-I, a new steady-state pHo is attained when the increase in cellular H+ efflux becomes balanced by an increase in paracellular H+ diffusion. Alternatively, the new steady-state pHo may arise when the decrease in pHo inhibits further proton efflux. In this regard, it has been shown that lowering pHo reduces net H+ efflux via Na+/H+ exchange and inhibits production of lactic acid by cultured calvaria (13, 19).The IGF-I receptor mediates IGF-I-induced increase in proton efflux. Our observations indicate involvement of the IGF-I receptor in mediating the rapid increase in proton efflux from osteoblast-like cells. The magnitude of this increase was dependent on the concentration of IGF-I. The EC50 for this response of 300 pM agrees well with the EC50 for responses mediated by the IGF-I receptor in other systems but is considerably lower than the EC50 for responses to IGF-I mediated through the insulin receptor (29). Furthermore, IGF-II (which activates the IGF-I receptor) caused an increase in proton efflux qualitatively similar to that induced by IGF-I. The smaller magnitude of the response to IGF-II is in keeping with the relative effects of IGF-I and IGF-II in other bone systems (8, 15, 18). Although both IGF-I and IGF II receptors are present on osteoblasts, the IGF-II receptor (the mannose 6-phosphate receptor) is not thought to mediate transmembrane signaling (8, 9). Thus the IGF-I receptor appears to mediate the effects of IGFs on proton efflux.
The increase in proton efflux and the associated decrease in pHo were sustained in the continued presence of IGF-I. Interestingly, washout of IGF-I was followed by recovery of both proton efflux and pHo to basal levels over a period of ~1 h. This time course is similar to that reported in rat fibroblasts for recycling of IGF-I receptors after their activation and internalization (31). In our system, a second comparable response to IGF-I could be elicited after a 1-h recovery. Taken together, these findings indicate that the acidification responses are sustained in the continued presence of IGF-I, are reversible upon washout, and do not exhibit short-term desensitization. Monitoring proton efflux may offer a convenient approach for studying the kinetics and regulation of desensitization to IGF-I and other ligands in osteoblasts and other cell types.Signaling pathways and metabolic mechanisms underlying the effect of
IGF-I on proton efflux.
The IGF-I receptor is an
2
2-heterotetrameric
receptor tyrosine kinase. Upon activation, the receptor
autophosphorylates and also phosphorylates intracellular substrates
such as the insulin receptor substrates and Shc. Several signaling
pathways are then stimulated, including those activated by
phospholipase C-
, Ras/MAP kinase, and PI 3-kinase, a
regulator of cellular metabolism. We investigated the role of PI
3-kinase in mediating the effects of IGF-I on proton efflux by use of
pharmacological probes. Wortmannin is a potent inhibitor of PI
3-kinase, with little or no effect on PI 4-kinase, Src
protein tyrosine kinase, protein kinase C, cAMP-dependent protein
kinase, cGMP-dependent protein kinase, or calmodulin-dependent protein
kinase II. However, wortmannin does inhibit phospholipase
A2, mitogen-activated protein
kinase, and myosin light-chain kinase, although markedly higher
concentrations of wortmannin are required to inhibit the latter two
enzymes than are required to inhibit PI 3-kinase (10, 25). In our
study, we found that the effects of IGF-I on proton efflux were
virtually abolished by wortmannin (100 nM), a concentration that
selectively inhibits PI 3-kinase in other intact cell systems. The
specificity of wortmannin was investigated using PTH, which signals
through both adenylyl cyclase and phospholipase C-
. In UMR-106
cells, PTH enhances proton efflux through elevation of cAMP (S. J. Harvey and S. J. Dixon, unpublished data). The lack of effect of
wortmannin on the increase in proton efflux induced by PTH argues
against a nonspecific effect of this inhibitor on metabolic acid production.
Possible physiological significance of proton efflux from osteoblastic cells. Acid production by cells of the osteoblast lineage may serve several functions. Small changes in pHo modify gap junctional communication (30) and expression of egr-1 and type 1 collagen (11) in cultured osteoblasts. When osteoblasts are actively synthesizing and secreting osteoid in vivo, it is possible that the pHo of the microenvironment between the osteoblast layer and the mineralizing front is regulated, affecting both the rate of mineral formation and phase transformation. An acidic zone beneath the active osteoblast layer may prevent mineralization of the osteoid seam during bone formation.
It is also possible that acid production by bone-lining cells and adjacent osteogenic cells activates osteoclastic bone resorption (1, 2, 7, 26). In vitro studies of pit formation by rat osteoclasts have shown that there is little, if any, resorptive activity at values of pHo >7.3. Slight decreases in pHo markedly stimulate osteoclastic resorption, which is maximally active at pHo values <7.0 (2). In our system, the compartment beneath confluent osteoblasts was acidified to a steady-state value 0.2-0.3 pH units below the pH of the superfusion medium. Furthermore, in response to IGF-I, osteoblasts acidified this compartment by an additional 0.05-0.1 pH units. Thus it is possible that localized acidification contributes to the stimulatory effects of IGF-I on resorption in vitro and in vivo. However, it is difficult to estimate the actual value of the local pHo changes in vivo, which will depend on proton efflux, buffering power, volume of the compartment, and permeability of the cell layer. The buffering power of extracellular fluid in vivo is greater than that of the superfusion media used in vitro. This would tend to make the changes in pHo in vivo smaller than those seen in vitro. On the other hand, the volume of the fluid compartment between the osteogenic cell layer and the bone matrix in vivo is likely very small, tending to enhance pHo changes in vivo. Although little is known about the permeability characteristics of the osteoblast layer in vivo, the presence of a "functional membrane or cellular envelope, which separates the extracellular fluids of bone from those of the animal as a whole" has been suggested by several authors (19). Like IGF-I, PTH is thought to stimulate osteoclastic resorption indirectly through a primary effect on osteoblastic cells. It is noteworthy that PTH enhances proton efflux from UMR-106 cells (Fig. 8) and SaOS-2 human osteoblast-like cells (4). Thus local acidification may contribute to the resorptive effects of both IGF-I and PTH. It will be of interest to determine whether other resorptive agents also stimulate acid efflux from osteoblasts and to assess the contribution of local changes in pHo to the regulation of osteoclastic resorption in vivo. In conclusion, IGF-I enhances acid production by osteoblastic cells via a signaling pathway dependent on activation of PI 3-kinase. The ensuing drop in pHo may then modulate important processes such as mineralization and osteoclastic resorption. In this regard, wortmannin has been shown to inhibit bone resorption in vitro and in vivo (23). It is possible that, in addition to direct effects on osteoclasts, wortmannin also inhibits resorption indirectly through suppression of acid production by osteoblasts. ![]() |
ACKNOWLEDGEMENTS |
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We thank Elizabeth Pruski for expert technical assistance with cell culture and glucose uptake studies, Harry Leung (Dept. of Zoology, Univ. of Western Ontario) for help with scanning electron microscopy, Dr. Graeme K. Hunter (School of Dentistry, Univ. of Western Ontario) for use of the autotitrator system, and Dr. Stephen M. Sims (Dept. of Physiology, Univ. of Western Ontario) for helpful comments and suggestions.
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FOOTNOTES |
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These studies were supported by the Medical Research Council of Canada (MRC). A. Santhanagopal is the recipient of an MRC Studentship.
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.
1 These procedures were approved by the Council on Animal Care of The University of Western Ontario.
2 Furthermore, the rapid initial drop in pHo introduced an artifact, which caused an apparent enhancement of the initial increase in proton efflux induced by IGF-I.
Address for reprint requests and other correspondence: S. J. Dixon, Dept. of Physiology, Faculty of Medicine and Dentistry, The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: jdixon{at}physiology.uwo.ca).
Received 20 January 1999; accepted in final form 27 April 1999.
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