INTRODUCTION

The parathyroid hormone/parathyroid hormone-related protein receptor (PTHR)¹ is a member of the G protein-coupled, seven-transmembrane domain superfamily of receptors (1). The principal physiological action of PTH is on the regulation of Ca²⁺/P_i homeostasis, which is mediated by the PTHR in kidney and bone. The biochemical and molecular mechanisms of the receptor-mediated actions of PTH are not completely understood. PTH stimulates osteoclast-mediated bone resorption indirectly via the activation of the PTHR on osteoblasts (2); however, the mechanism coupling these events is not known. Additionally, PTH has an anabolic effect on bone when administered intermittently (3). It has been proposed that the diverse actions of PTH may be a consequence of activation of both the cAMP/PKA and inositol lipid/Ca²⁺/PKC signaling pathways by the PTHR in osteoblasts (4). Receptor regulation is also important in the action of PTH on osteoblasts (4-9). Homologous desensitization of PTH-stimulated increases in intracellular cAMP accumulation ([cAMP]_i) and in cytosolic free calcium concentration ([Ca²⁺]_i) and down-regulation of the PTHR have been described in several target cell systems (4-9).

Previous investigations of the physiological actions of PTH have largely used animal and whole bone experimental systems (10-14), while studies of PTHR signaling have concentrated on monitoring the activation of either adenylyl cyclase and the cAMP/PKA or phospholipase C and inositol lipid/Ca²⁺/PKC signaling pathways in osteoblastic cells in culture (4-9, 15). Microphysiometry is a novel method for monitoring cellular metabolism (16) and has been effectively adapted to monitor acid secretion and metabolic rates in small populations of cultured cells in real time (17, 18). Because local pH homeostasis is physiologically important in mineral ion metabolism, and PTH-stimulated bone resorption has been linked to acid production by osteoclasts (13), we utilized microphysiometry to monitor the effect of PTH on extracellular acidification in osteoblasts, the primary target cell for PTH in bone.

The Cytosensor microphysiometer utilizes silicon-based, light-addressable potentiometric sensors to make rapid, precise determinations of extracellular pH. The Cytosensor monitors the rate at which cells secrete the acidic byproducts of metabolism, the extracellular acidification rate (ECAR). Cells acidify the environment via ion exchangers and pumps such as the Na⁺/H⁺ antiporter and the H⁺-ATPase and by transporting metabolites such as lactic acid across the plasma membrane (19). Several seven-transmembrane G protein-coupled receptors such as adrenergic, dopamine, and muscarinic receptors elicit concentration-dependent increases in ECAR in response to agonist binding (17, 18, 20); however, the physiological significance of receptor-mediated stimulation of extracellular acidification in these cell systems is not clear. In contrast, acidification of the bone microenvironment is known to be functionally important in skeletal metabolism and Ca²⁺/P_i homeostasis (13). Osteoclast-mediated bone resorption depends on the accumulation of acid at the site of resorption (13, 21, 22), and small fluctuations in extracellular pH in bone affect osteoclastic bone resorption (23-25). Stimulation of bone resorption by PTH is also linked to increased glycolytic acid production (13), but the cellular and molecular mechanisms of this action of the hormone are unknown. For these reasons, we utilized microphysiometry to demonstrate that agonist activation of the PTHR stimulates a large, acute, dose-dependent increase in ECAR, which we have utilized for a new analysis of PTHR signaling and regulation in osteoblastic cells.

EXPERIMENTAL PROCEDURES

Culture media and sera were obtained from Life Technologies, Inc. (Grand Island, NY). hPTH-(1-34) and human vasoactive intestinal peptide (VIP) were obtained from Peninsula Laboratories (Belmont, CA). [Nle^8,18,D-Trp¹²,Tyr³⁴]bPTH-(7-34)NH₂ was a generous gift from Dr. Michael Chorev (Beth Israel Hospital, Boston, MA). Phorbol 12-myristate 13-acetate (PMA), thapsigargin, and nigericin were purchased from Calbiochem (La Jolla, CA), and Ro-20-1724 was obtained from Biomol (Plymouth Meeting, PA). Fura-2, acetoxymethyl ester (Fura-2/AM) and 2,7-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein, acetoxymethyl ester (BCECF/AM) were supplied by Molecular Probes, Inc (Eugene, OR). 1-O-Hexadecyl-2-O-methyl-rac-glycerol (AMG-C₁₆), 8-bromo-cyclic AMP (8-Br-cAMP), ()-isoproterenol, and additional chemicals were purchased from Sigma. The Cytosensor microphysiometer and supplies were obtained from Molecular Devices (Sunnyvale, CA). Tissue culture plasticware was purchased from Beckton Dickinson (Lincoln Park, NJ).

Culture of the cells used in this study has been described previously. In brief, human osteoblast-like SaOS-2 (26-28) and rat osteoblast-like UMR-106 (29) cells were grown as monolayers in minimal essential medium supplemented with 5% horse serum and 5% fetal bovine serum (MEM⁺). Rat pituitary GH₄C₁ (30) and F₄C₁ (31) cells were cultured as monolayers in Ham's F-10 nutrient mixture supplemented with 15% horse serum and 2.5% fetal bovine serum (Ham's F-10⁺). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. The cells were subcultured 1:5 every fifth day or as necessary for experiments.

Experiments were performed as detailed previously (17, 18, 20). Briefly, 12-24 h before an experiment, cells were subcultured onto the polycarbonate membranes of transwell capsule cups (Corning Costar, Cambridge, MA) in the following manner. Cells were detached from the plastic culture dish with 0.5 mg/ml trypsin and then suspended in MEM⁺, and the concentration of cells in suspension was determined. The cell suspension was diluted to a concentration of 5 × 10⁵ cells/ml (1 × 10⁶ cells/ml for GH₄C₁ cells), and 1 ml of the suspension was seeded into each transwell capsule cup placed in a 12-well culture plate. The cells were then incubated at 37 °C until the beginning of the experiment. Immediately preceding an experiment, the low buffered MEM running buffer (MEM lacking NaHCO₃ (to minimize buffering capacity), supplemented with 30 µM H₃CCO₂H (the PTH vehicle) and 0.1% bovine serum albumin or 5% horse serum (carrier proteins)) was prepared. The Cytosensor system was warmed to 37 °C and flushed with running buffer. The transwell capsule cups containing the cells were fitted with a spacer gasket and capsule cup insert to create a microvolume flow chamber containing the cells. The assembled capsule cups were then placed on the sterile silicon sensors and mounted on the Cytosensor. The Cytosensor was programmed to perfuse the cells with running buffer at a rate of approximately 100 µl/min for periods of 68 s interrupted by 22-s periods of no flow. The pH of the extracellular media was measured at 1-s intervals, and the rate of acidification was determined by calculating the least squares fit to the slope of the pH profile while the flow of buffer was stopped. After being loaded into the Cytosensor, cells were allowed to equilibrate until a stable base-line ECAR was reached, and then they were exposed to pharmacological and agonist agents as described under "Results." The low buffered, balanced salt solutions (BSS) used in experiments to control Na⁺/H⁺ exchange consisted of 0.3 mM CaCl₂, 0.6 mM MgCl₂, 0.5 mM KH₂PO₄, 3 mM KCl, 5 mM D-glucose, and 130 mM either C₅H₁₄ClNO or NaCl.

For the purpose of comparing ECARs from many different experiments, microphysiometer data were normalized utilizing Cytosoft software (Molecular Devices, Sunnyvale, CA). A minimum of five consecutive data points from the base-line ECAR of each cell population were selected and averaged, and then all other data points were expressed as a percentage of this basal average.

SaOS-2 cells were prepared for analysis of pH_i and [Ca²⁺]_i as described previously (32, 33). In brief, the cells in the monolayer were detached from the culture dish using Ca²⁺-free Hanks' balanced salt solution (HBSS) consisting of 120 mM NaCl, 4 mM KCl, 10 mM D-glucose, and 20 mM HEPES, pH 7.2, supplemented with 5 mM EDTA. The cells were then washed three times and resuspended in HBSS containing 1 mM CaCl₂ at a concentration of approximately 2 × 10⁶ cells/ml. Fura-2/AM or BCECF/AM was added to the suspension to a final concentration of 1 or 5 µM, respectively. The suspension was incubated at 37 °C in the dark with gentle shaking for 60 min. After incubation, the cells were washed three times in HBSS containing 1 mM CaCl₂ and then two times with low buffered BSS and resuspended in the low buffered BSS at a concentration of 5 × 10⁶ cells/ml. The suspension was loaded in a UV grade acrylic cuvette (Spectrocell, Oreland, PA) in a Hitachi F-2000 spectrofluorometer (Rye, NH) warmed to 37 °C and stirred constantly. [Ca²⁺]_i was determined using the ratio of Fura-2 emission fluorescence at 510 nm at excitation wavelengths of 340 and 380 nm as described previously (34). pH_i was determined by monitoring the fluorescence emission of BCECF at 535 nm at an excitation wavelength of 500 nm.

Each spectrofluorometric experiment shown was repeated at least three times with qualitatively and quantitatively similar findings.

RESULTS

Basal ECAR in serum-free, low buffered MEM was 47 ± 15 µV/s (mean ± S.D., n = 55) when 5 × 10⁵ cells were seeded in each transwell capsule cup. Human PTH-(1-34) elicited an acute increase in ECAR (Fig. 1). The maximum PTH-stimulated increase in ECAR was 68 ± 24 µV/s (mean ± S.D., n = 55) above basal, a 2.4-fold increase above the resting acidification rate, and the EC₅₀ was about 2 nM (Fig. 2). The peak in ECAR occurred within 45 s of agonist addition and usually returned to basal levels within 5 min even in the persistent presence of PTH (Fig. 1). In some experiments, the ECAR dropped below the original basal level or remained slightly above it following a PTH exposure. Such modest variations in the pattern of ECAR responses to agonists have been reported in analogous studies in other cell types (18). Similar, acute responses were also elicited by bPTHrP-(1-34)NH₂ (data not shown). These results demonstrate that PTH elicited large, acute, dose-dependent increases in ECAR in SaOS-2 cells. The specificity of the ECAR response was investigated in the following experiments.

Human osteoblast-like SaOS-2 cells, which endogenously express the PTHR, and rat pituitary F₄C₁ and GH₄C₁ cells, which do not express the PTHR,² were examined. Each cell type was incubated with 100 nM hPTH-(1-34) for 2 min, and ECAR was monitored (Fig. 3). PTH induced an increase in ECAR in SaOS-2 cells, but no response was observed in the two PTHR-deficient cell strains. F₄C₁ and GH₄C₁ cells were responsive to an appropriate peptide hormone in the Cytosensor system, because thyrotropin-releasing hormone, for which these cells express the receptor, induced large increases in ECAR in these two cell types (data not shown). In addition, hPTH-(1-34) stimulated rapid increases in ECAR in rat osteoblast-like UMR-106 cells, which also express the PTHR (data not shown). These results suggest that the PTH-induced increase in ECAR is mediated by the PTHR. Additional evidence to support this conclusion is given below.

SaOS-2 cells were incubated with the PTHR antagonist [Nle^8,18,D-Trp¹²,Tyr³⁴]bPTH-(7-34)NH₂ (35). A 25-min pretreatment of cells with 200 nM [Nle^8,18,D-Trp¹²,Tyr³⁴]bPTH-(7-34)NH₂ elicited no increase in ECAR, and it reduced the ECAR response elicited by 20 nM hPTH-(1-34) to about 25% of the control value, while the response of the cells to 20 nM VIP was unaffected (Fig. 4). The inhibition of the PTH-induced increase in ECAR by [Nle^8,18,D-Trp¹²,Tyr³⁴]bPTH-(7-34) was reversed by washing the cells with buffer for 90 min (data not shown). A 25-min preincubation of SaOS-2 cells with 100 nM [Nle^8,18,D-Trp¹²,Tyr³⁴]bPTH-(7-34)NH₂ caused a shift in the ECAR dose-response curve for hPTH-(1-34) about 10-fold to the right, increasing the EC₅₀ to about 10 nM (Fig. 5). These results demonstrate that the PTH-induced ECAR response is mediated by the PTHR in SaOS-2 cells. The following experiments were designed to investigate the response of osteoblast-like cells to repeated exposures to PTH.

Microphysiometry was used to analyze desensitization of the receptor-mediated, PTH-induced ECAR response. Because the increase in ECAR induced by PTH was acute in nature and the acidification rates of the cells returned rapidly to the basal level, it was possible to monitor repeated sequential responses to PTH in real time. An initial response to a 2-min challenge with 100 nM hPTH-(1-34) was followed by a refractory period of approximately 15 min during which the response of the same cells to a second challenge with 100 nM hPTH-(1-34) was reduced relative to the first response. When two 2-min agonist treatments were separated by a 2.5-min wash, the second PTH-induced response was reduced to about 13% of the first (Fig. 6). As the wash time between treatments was increased, the magnitude of the second response to PTH increased, with nearly full recovery after 14.5 min of hormone-free perfusion (Fig. 6). Interestingly, decreased responsiveness after the initial exposure to PTH was not progressive. In cells treated with 100 nM hPTH-(1-34) for 2-min intervals separated by 7-min washes, the first response to hPTH-(1-34) was the largest, and the second response was reduced to about 50% of the first response; however, no further reduction in responsiveness was observed in up to nine subsequent, sequential agonist exposures (data not shown).

The effect of continuous long term agonist exposure on the acute PTH-induced ECAR response was also investigated. In preparation for these experiments, SaOS-2 cells were subcultured into capsule cups as described under "Experimental Procedures," and for 13 h prior to the experiment, the media covering the cells was changed to MEM⁺ supplemented with 100 nM hPTH-(1-34) or to control MEM⁺. Immediately before the experiments, the cells were quickly washed three times and then placed on the Cytosensor and perfused with hormone-free running buffer for about 45 min, until a stable basal acidification rate was achieved. The cells were then sequentially exposed to increasing concentrations of hPTH-(1-34) over the course of 3 h. The acute maximal response of PTH-pretreated cells to fresh agonist exposure was reduced to about 50% of the maximal response of control cells; however, the response of the control and pretreated cells to 100 nM VIP treatment was the same (data not shown). The 3-4-h lag between the washout of the PTH pretreatment and the completion of the measurement of ECAR dose-response could have allowed the preincubated cells to recover some responsiveness to PTH; nevertheless, a significant (p < 0.05) reduction in responsiveness to hormone exposure was measured. The mechanism of the PTH-stimulated increase in ECAR was then examined.

When SaOS-2 cells were incubated for 30 min with 1 mM 8-Br-cAMP, there was no large, acute increase in ECAR such as that elicited by PTH in the same cells; only a transient 5-20% increase in ECAR was observed (Fig. 7). When UMR-106 cells were exposed to 1 mM 8-Br-cAMP, a persistent increase in ECAR resulted (data not shown). The increase in ECAR in UMR-106 cells stimulated by 8-Br-cAMP peaked at 1.7-fold above basal levels and returned to basal levels only after a 50-min wash, indicating that this cAMP analog was active in the Cytosensor system.

The effect of a high concentration of exogenous cAMP on the PTH-induced increase in ECAR was then examined in SaOS-2 cells. Cells were pretreated for 30 min with either 1 mM 8-Br-cAMP, clamping the intracellular cAMP concentration at supramaximal levels above those generated by PTH exposure, or control running buffer, and then the cells were exposed to 10 nM hPTH-(1-34) for 2 min. The PTH-induced ECAR response in SaOS-2 cells was not affected by the simultaneous presence of 1 mM 8-Br-cAMP (Fig. 7). SaOS-2 cells were also incubated with 100 nM forskolin to stimulate endogenous cAMP production, but no acute increase in ECAR was observed (data not shown).

Finally, SaOS-2 Ca#4A cells were used to analyze further the role of the cAMP/PKA signaling pathway in the PTH-stimulated increase in ECAR. SaOS-2 Ca#4A cells are a subclone of SaOS-2 cells in which enhancement of cAMP formation does not activate PKA due to the overexpression of a mutant PKA regulatory subunit (36). hPTH-(1-34) (100 nM) stimulated an acute increase in ECAR in these cells, while 1 mM 8-Br-cAMP did not produce any increase in ECAR, demonstrating that the cAMP-resistant phenotype was being expressed (data not shown). Based on the results of all of the above experiments, we conclude that the increase in ECAR elicited by PTH does not depend on receptor-mediated stimulation of adenylyl cyclase, an increase in [cAMP]_i, or activation of PKA.

Previous studies of PTHR signaling in SaOS-2 cells did not detect acute increases in [Ca²⁺]_i in response to PTH using single wavelength fluorescence monitoring (33). However, in other osteoblastic cell strains, PTHR occupancy has been linked to activation of the inositol lipid signaling pathway (4, 5, 15). In the current study, a subclone of wild-type SaOS-2 cells (SaOS-2 #95) was used. In this clone, PTH did induce acute increases in [Ca²⁺]_i as monitored by using the Ca²⁺-sensitive fluorescent probe Fura-2 and dual wavelength spectrofluorometric analysis, but all other aspects of this clone appear to be the same as wild-type SaOS-2 cells (data not shown). hPTH-(1-34) (100 nM) caused an acute increase above basal level in [Ca²⁺]_i of 70 ± 10 nM (mean ± S.E., n = 11) (Fig. 8A). Pharmacological depletion of intracellular calcium stores with 1 µM thapsigargin elicited increases in [Ca²⁺]_i of 142 ± 9 nM (mean ± S.E., n = 18), and the PTH-induced increase in [Ca²⁺]_i was abolished by thapsigargin pretreatment (Fig. 8B). Thapsigargin (1 µM) induced a modest and somewhat prolonged increase in ECAR, but depletion of thapsigargin-sensitive Ca²⁺-stores did not reduce the PTH-stimulated increase in ECAR (Fig. 8C), demonstrating that the acute PTH-induced increase in ECAR does not involve the release of Ca²⁺ from thapsigargin-sensitive (inositol 1,4,5-triphosphate-sensitive) intracellular Ca²⁺ stores.

We next examined the effect of AMG-C₁₆, a PKC inhibitor that has been shown to have little effect on PKA or Ca²⁺/calmodulin-dependent protein kinase (37), on the PTH-induced increase in ECAR. AMG-C₁₆ (200 µM) reduced the increase in ECAR elicited by 20 nM PMA to 35% of the control PMA response (data not shown) and reduced the response to 100 nM hPTH-(1-34) to 37% of the control PTH response, while the increase in ECAR stimulated by 1 mM ()-isoproterenol was unaffected (Fig. 9). In addition, SaOS-2 cells were pretreated with PMA (100 nM) for 24 h, which down-regulates PKC expression in these cells (38), and then exposed to 100 nM PTH. Down-regulation of PKC resulted in a 73% decrease in the PTH-stimulated increase in ECAR (Fig. 10) and a reduction of about 50% in a subsequent PMA-stimulated increase in ECAR (data not shown). Taken together, these results indicate that activation of PKC is involved in the PTH-stimulated increase in ECAR in SaOS-2 cells.

It has been demonstrated that the increase in ECAR stimulated by some G protein-coupled receptors is mediated by activation of Na⁺/H⁺ exchange (20, 39). The PTHR has been shown to modulate the activity of Na⁺/H⁺ exchange in certain osteoblast-like cell lines (40-42). Therefore, we equilibrated SaOS-2 cells in either Na⁺-containing, low buffered BSS or Na⁺-free, low buffered BSS, which inhibits Na⁺/H⁺ exchange. The PTH-stimulated ECAR response was equal in Na⁺-free and Na⁺-containing buffer (Fig. 11). To ensure that the Na⁺-free BSS effectively inhibited Na⁺/H⁺ exchange, SaOS-2 cells were loaded with the pH-sensitive fluorescent probe BCECF/AM and then acidified with 1 µM nigericin (a K⁺/H⁺ ionophore). In nigericin-acidified SaOS-2 cells, PMA induces alkalinization via a Na⁺/H⁺ exchanger-dependent mechanism (42). This PMA-induced alkalinization was completely blocked in Na⁺-free buffer (data not shown). Furthermore, the large, acute ECAR response of GH₄C₁ cells to the neuropeptide agonist thyrotropin-releasing hormone was abolished in the Na⁺-free buffer (data not shown). These results demonstrate that the PTH-stimulated increase in ECAR is independent of Na⁺/H⁺ exchange in SaOS-2 cells.

DISCUSSION

Receptor-mediated action of PTH on osteoblasts is essential in the maintenance of Ca²⁺/P_i homeostasis, but the mechanisms by which PTH controls mineral metabolism are not completely clear. PTH can activate both the cAMP/PKA and inositol lipid/Ca²⁺/PKC signaling pathways (4-9, 15), but the relative, physiological importance of these individual pathways in the diverse actions of PTH on bone is not completely understood. Stimulation of the cAMP/PKA signaling pathway by PTH in osteoblasts has been linked to osteoclast-mediated bone resorption (43, 44); however, the precise biochemical link between osteoblasts and osteoclasts in bone resorption has not been elucidated. PTH also has an anabolic effect on bone when administered intermittently, but the molecular mechanisms of this action are not understood (3). Additionally, acid production and pH homeostasis in the bone microenvironment are important in regulation of mineral metabolism and bone resorption (23-25), and PTH action has been linked to acid production in resorbing bone (13); therefore, the data presented here, demonstrating that PTH stimulates ECAR in human osteoblast-like SaOS-2 cells, has important consequences in bone physiology and mineral metabolism.

Early investigations of mineral metabolism linked acid production by glycolysis to stimulation of bone resorption mediated by PTH (13). In vitro exposure of whole bones to PTH resulted in the production of lactate and citrate (13, 45-47), and local pH is important in controlling bone mineral content (23-25). Activated osteoclasts concentrate acid in the resorptive compartment, resulting in the solubilization of Ca²⁺ and P_i from hydroxyapatite crystals, allowing for subsequent degradation of matrix proteins by proteases (13, 21, 22). In addition, in vivo and in vitro studies show that decreased pH results in physiochemical and osteoclast-mediated release of Ca²⁺ from bone (23, 25). Although osteoblasts are the primary cellular target for PTH in bone and local pH homeostasis is known to be important, evidence is lacking concerning the role of osteoblasts in the production of acid in the bone environment. Based on the findings described in this report, we conclude that PTH directly enhances acid production by osteoblasts. This effect occurs in addition to osteoclast secretion of acid, which is an indirect action of PTH.

We investigated the signaling mechanisms involved in stimulation of increases in ECAR by PTH in SaOS-2 cells. PTH causes acute increases in cAMP accumulation in these cells (7, 8); therefore, the role of the cAMP/PKA signaling pathway in the PTH-induced increase in ECAR was examined. Activation of PKA with 8-Br-cAMP produced a small increase in ECAR, which did not mimic stimulation of ECAR by PTH either temporally or quantitatively. It is possible that the inability of 8-Br-cAMP to mimic the large, acute PTH-induced increase in ECAR was due to slow diffusion of the analog into cells relative to the acute increase in [cAMP]_i stimulated by PTH. However, the PTH-induced increase in ECAR was not altered by clamping [cAMP]_i at supramaximal concentrations by preincubation with 8-Br-cAMP. In addition, increases in ECAR induced by submaximal concentrations of PTH were neither augmented nor abrogated by the presence of 8-Br-cAMP (data not shown). If the PTH-induced increase in ECAR was the result of a receptor-mediated increase in intracellular cAMP, the presence of high concentrations of 8-Br-cAMP would be expected to abrogate subsequent receptor-mediated increases in ECAR as seen in other receptor-mediated increases in ECAR that are dependent on increases in [cAMP]_i (20, 48). Furthermore, PTH was as efficacious in stimulating an acute increase in ECAR in cAMP-resistant SaOS-2 Ca#4A cells as it was in wild-type SaOS-2 cells, and forskolin treatment, which increased [cAMP]_i, did not induce an increase in ECAR. Finally, the phosphodiesterase inhibitor Ro-20-1724 was used to elevate basal [cAMP]_i, but no potentiation of the PTH-induced increase in ECAR resulted (data not shown). Other cAMP-dependent actions of PTH are known to be potentiated by inhibition of phosphodiesterase activity. Based on these data collectively, we conclude that activation of the cAMP/PKA signaling pathway is not involved in the acute, PTH-stimulated increase in ECAR in SaOS-2 cells.

Previous studies on SaOS-2 cells reported that PTH did not stimulate acute increases in [Ca²⁺]_i (33). However, PTH has been shown to elicit transient increases in [Ca²⁺]_i and generate inositol polyphosphates in other osteoblast-like cell lines (4, 5, 15); therefore, we examined the role of the inositol lipid/Ca²⁺/PKC signaling pathway in the stimulation of ECAR by PTH. Using dual wavelength spectrofluorometry, transient increases in [Ca²⁺]_i were measured in the subclone of SaOS-2 cells used in the investigations presented in this report. Heterogeneity of intracellular calcium responses to PTH in osteoblasts has been reported (49, 50). Therefore, we believe that we isolated a subpopulation of SaOS-2 cells that does respond to PTH with an increase in [Ca²⁺]_i, allowing us to consider the contribution of this signaling event to stimulation of increased ECAR elicited by PTH. The increases in [Ca²⁺]_i were dependent on the release of Ca²⁺ from intracellular stores, because pharmacological depletion of these pools with thapsigargin blocked subsequent PTH-stimulated increases in [Ca²⁺]_i. The PTH-induced increase in ECAR, however, was not blocked by thapsigargin pretreatment. PTH also stimulated a similar, acute increase in ECAR, but not [Ca²⁺]_i, in the wild-type SaOS-2 cells from which the subclone used in this study was derived (data not shown), indicating that the increase in ECAR was not dependent on the transient increase in [Ca²⁺]_i. We then investigated the role of PKC in the PTH-induced increase in ECAR. Treatment of cells with the PKC inhibitor AMG-C₁₆ significantly reduced the PTH-stimulated increase in ECAR. In control experiments, AMG-C₁₆ inhibited the PMA-induced increase in ECAR, while it did not decrease basal ECAR or affect stimulation of ECAR by isoproterenol, indicating that AMG-C₁₆ was inhibiting PKC and was not nonspecifically toxic to the cells. Additionally, pretreatment of SaOS-2 cells with PMA resulted in a substantial reduction in the PTH-induced increase in ECAR. Pretreatment of SaOS-2 cells with PMA down-regulates PKC (38), and it reduced PMA-stimulated increases in ECAR to about 50% of control (data not shown), demonstrating the specificity of the effect of the pretreatment on PKC. Taken together, these results indicate that stimulation of ECAR by PTH is dependent on the activity of PKC but independent of transient increases in [Ca²⁺]_i, demonstrating that PTHR activation of the inositol lipid/PKC signaling pathway may be functionally relevant to osteoblast physiology.

Receptor-mediated increases in ECAR have been linked to Na⁺/H⁺ exchange in several systems (20, 39), and proton extrusion by these exchangers could account for acute acidification of the extracellular environment. In addition, in vitro experiments have suggested that PTH can modulate Na⁺/H⁺ exchangers via multiple signaling pathways (40-42, 51). To determine the contribution of Na⁺/H⁺ exchange in the PTH-induced increase in ECAR, SaOS-2 cells were incubated in Na⁺-free buffer, which blocks Na⁺/H⁺ exchange, and then exposed to PTH. The PTH-induced increase in ECAR was not reduced relative to control in Na⁺-free buffer. In addition, the Na⁺/H⁺ exchanger inhibitor, 5-(N-methyl-N-isobutyl) amiloride, did not inhibit the PTH-induced increase in ECAR (data not shown), indicating that stimulation of ECAR by PTH was independent of Na⁺/H⁺ exchange in SaOS-2 cells.

Desensitization of the PTHR-mediated increase in ECAR was also investigated. The PTH-stimulated increase in ECAR peaked within 45 s of PTH addition and returned rapidly to basal levels. The acute nature of the increase allowed us to monitor the ECAR response of cells to repetitive exposures to hormone on a time scale of minutes. Acute, PTH-induced increases in ECAR were followed by a relative refractory period of about 15 min. During this period, the cells responded less vigorously, relative to the first response, when rechallenged with hormone. Thus, the ECAR response to PTH was rapidly attenuated, but the cells quickly recovered responsiveness after agonist exposure was terminated. The acute nature of the ECAR response and the rapid recovery of the ability to respond suggest that the PTHR and/or the signaling pathway that it stimulates is under tight regulation. The results presented in this report demonstrate that stimulation of ECAR by PTH involves activation of PKC. Prolonged pharmacological activation of PKC by PMA results in an extended increase (over many minutes to hours) in ECAR in SaOS-2 cells (data not shown), suggesting that the PTH-induced increase in ECAR is not limited by the transient capacity of PKC to stimulate increased ECAR; thus, the PTH-induced response may be regulated upstream of the activation of PKC. Regulation at the level of the receptor could explain the acute nature of the increase in ECAR stimulated by PTH. This regulation may involve a mechanism such as that which has been shown to regulate signaling by the -adrenergic receptor system by uncoupling the activated receptor from G proteins through the action of G protein-coupled receptor kinases and arrestins (52-54).

The functional significance of the acute ECAR response in osteoblasts is not yet known. It is not straightforward to test for significance in intact skeletal tissue in vitro or in animals in vivo, because, as is now known, both osteoblasts and osteoclasts extrude acid in response to PTH. Until more is understood about the mechanisms in each cell type, there is no known way of selectively inhibiting acid production in one of these cell types to determine the physiological consequence. Nevertheless, due to the temporal link of the two effects, it is conceivable that the acute ECAR response in osteoblasts is, in some unknown way, linked to the anabolic response of PTH in bone that occurs after transient, but not persistent, exposure of animals and humans to PTH (3).

In summary, we have demonstrated that PTH stimulates an acute, receptor-mediated, dose-dependent increase in ECAR in human osteoblast-like SaOS-2 cells. The ECAR increase is independent of the cAMP/PKA signaling pathway, transient increases in [Ca²⁺]_i, and Na⁺/H⁺ exchange, but it is dependent on the activity of PKC. The PTH-stimulated increase in ECAR is rapidly, but transiently, desensitized; and long term pretreatment with PTH results in reduced ECAR responses.