Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2′-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.
Previously, we used microphysiometry to examine the signal transduction pathway utilized by parathyroid hormone (PTH) to elicit an acute increase in the extracellular acidification rate (ECAR) in human osteoblast-like SaOS-2 cells.(1) This method also was employed by Bodine and Komm to show the presence of osteocalcin receptors on bone-derived cells and by others to study G protein–coupled receptor-mediated signaling events in several cell systems.(2–4) Ease of use and possible physiological relevance of acidification of the local extracellular environment in bone make this method an attractive tool for studying intact bone. In addition, using the whole tissue avoids ambiguities associated with use of isolated cells in primary culture or transformed cell lines. Here, we report a method of measuring ECAR responses to hormones in calvarial tissue taken directly from neonatal mice and an analysis of the signal transduction pathway for the PTH-induced change in ECAR in this tissue.
In response to hormone binding, the PTH receptor (PTH1R) can activate the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and inositol lipid/Ca2+/protein kinase C (PKC) signal transducing pathways and initiate the actions of PTH on bone.(5–10) Continuous exposure to high concentrations of PTH in animals or in organ culture leads to enhanced bone resorption, while intermittent administration of low doses of PTH in animals and people has an anabolic effect.(11,12) Catabolic responses occur when osteoblasts, stimulated by PTH, produce osteoclast differentiation factor, which then recruits osteoclasts to destroy bone.(13) The mechanism of the anabolic action of PTH is unclear, but it is under intense examination to find an effective treatment for severe osteoporosis. Development of PTH and PTH analogs as drugs requires assays that will distinguish between the PKC/inositol lipid and cAMP/PKA pathway-activating properties of these potential therapeutic agents.
Acidifying or alkalinizing the media used in calvarial organ culture modulates bone resorption and markers of bone growth, with low pH favoring resorption and higher pH favoring formation.(14,15) Changes as small as 0.1 pH unit have been shown to affect significantly osteoclastic bone resorption.(16) We have previously reported that human osteoblast-like SaOS-2 cells respond to PTH with an increase in ECAR, and that the acute change is mediated by the PKC signal transduction pathway and is not dependent on the cAMP/PKA pathway nor the Na+/H+ exchanger.(1) Here, we extend this approach, using microphysiometry to measure PTH-induced acidification responses in normal intact neonatal mouse calvaria.
Culture media were obtained from Media Tech (Herndon, VA, U.S.A.). Human PTH (hPTH) hPTH(1–34) and hPTH(53–84) were obtained from Bachem (Torrence, CA, U.S.A.). Bovine PTH (bPTH) analogs [Tyr34]bPTH(7–34)NH2 and [Nle8,18, Tyr34]bPTH(3–34)NH2 were purchased from Peninsula Laboratories (Belmont, CA, U.S.A.). Salmon calcitonin was obtained from Armour Pharmaceutical Company (Kankakee, IL, U.S.A.). Ethoxolamide was a generous gift of Dr. Curtis Conroy (University of Florida College of Medicine, Gainesville, FL). Forskolin, N6-2′-0-dibutyryl-adenosine 3′:5′-cAMP(db-cAMP), isobutylmethylxanthine (IBMX), acetazolamide, bovine serum albumin (BSA), and additional chemicals were purchased from Sigma (St. Louis, MO, U.S.A.). Thapsigargin (Tg) was obtained from Research Biochemicals International (Natick, MA, U.S.A.). Phorbol 12-myristate 13-acetate (PMA) was obtained from LC Services Corporation, (Woburn, MA, U.S.A.). Pregnant ICR mice and litters were obtained from Harlan (Indianapolis, IN, U.S.A.). The Cytosensor microphysiometer and supplies were purchased from Molecular Devices (Sunnyvale, CA, U.S.A.).
Calvaria were removed from decapitated 1- to 4-day-old mice weighing less than 2.5 g and immediately placed in 75% running buffer (Dulbecco's modified Eagle medium (DMEM) without NaHCO3 or pyruvate, containing 4.5 gm/liter glucose, 0.2% BSA, 30 μM acetic acid, and 25% phosphate-buffered saline (PBS). Using a sterile razor blade, a strip of calvarial tissue was cut measuring 2 mm × 7 mm containing the sagittal suture and adjacent tissue. This bone sample was placed in the chamber of the Cytosensor, perpendicular to the flow of media, midway between the inlet and outlet, and sandwiched between the capsule insert and transwell membranes using two 50-μm-thick spacers (see Ref. 3 for a detailed description of the instrumentation). The chamber volume was 2.8 μl. The external surface (scalp side) was placed closest to the sensor chip unless noted otherwise. Running buffer at 37°C was pumped constantly through the chambers at a rate of 0.15 ml/minute. (60% of maximum flow rate). Raw data (μV/s) were collected every second and analyzed versus time. The slope of this curve was calculated every 3 minutes for an interval of 140 s and plotted versus time as –dpH/dt. Calculations were made using the Cytosoft software (Molecular Devices, Sunnyvale, CA, U.S.A.) from the least squares fit to the slope of the pH profile. To calibrate the machine at the end of an experiment and to determine machine drift, the bones were removed, and buffers of various known pHs were run through the chambers. Machine drift was subtracted from the data, and an empirically determined conversion factor was used to convert microvolts per second into pH units per minute. Data were analyzed by analysis of variance (ANOVA) using Fisher's least significant difference (LSD) test where appropriate.
Neonatal (4- to 5-day-old) mouse calvaria were divided into symmetrical halves along the sagittal suture and were cultured as free-floating bones in DMEM supplemented with 15% heat-inactivated horse serum (Bio Whittaker, Walkersville, MD, U.S.A.) containing 10 U/ml heparin (Elkins-Sinn, Inc., Cherry Hill, NJ, U.S.A.). Incubation was performed at 37°C under an atmosphere of 50% O2, 5% CO2, and 45% N2. Calvaria were preincubated for 24 h before experimental treatments were begun. After the 24-h preincubation period, medium was removed and replaced with either fresh control medium or medium containing specific treatments. Bone resorption was determined by measuring the accumulation of 40Ca2+ in the culture medium 72 h after the addition of test agents.(5) The concentration of total calcium in the medium was measured in 400-μl samples by a calcium-selective electrode using a NOVA 7 + 7 automatic calcium analyzer (NOVA Biomedical, Waltham, MA, U.S.A.).
Acidification in neonatal calvaria is stimulated by PTH
Mouse calvarial bone surrounding the sagittal suture was monitored for changes in ECAR using the Cytosensor microphysiometer. After equilibration, the chambers stabilized with an upward drift between 0.0006 and 0.0034 pH units/minute that occurred without or with tissue in the capsules. The average drifts with and without bones were not significantly different. When incubated with 100 nM hPTH(1–34), the acidification rate decreased transiently during the first 3 minutes of exposure and then increased for as long as PTH was present (Fig. 1). The mean change in –dpH/dt for the first 3 minutes of PTH treatment was 0.0022 pH units/minute (n = 26), which was significantly different from the vehicle control value of 0.00023 pH units/minutes (n = 18; p < 0.002). Maximum –dpH/dt for 100 nM PTH was about 0.0054 pH units/minute, occurring after 12 minutes of treatment. After about 30 minutes, –dpH/dt declined to a constant rate slightly above basal for as long as PTH was present. The average rates for the last 12 minutes of PTH exposure for treatment and control were significantly different (p < 0.001; n = 4). When PTH was removed from the perfusion medium after 1 h, there was a prompt fall in ECAR (Figs. 1 A and 1B). Calibration at the end of the experiment with running buffer of various pH values showed a linear relationship between pH and the voltage response; 1 pH unit was equivalent to 44.7 mV (SD = 0.13; n = 5), thus, the 1-h treatment with 100 nM hPTH(1–34) caused the pH in the chamber to decrease 0.11 pH units below the control. The dose-response analysis for the effect of hPTH(1–34) to increase ECAR gave a half-maximal effective concentration (EC50) of about 15 nM (Fig.1A, inset). These results show that PTH elicited a dose-dependent increase in extracellular acidification in neonatal mouse calvaria. Salmon calcitonin also produced an increase in ECAR, but, unlike PTH, when treatment was discontinued ECAR did not decrease over the subsequent 30 minutes (Fig. 2).
Anatomical and peptide specificity of PTH-induced increase in acidification
The area surrounding the sagittal suture portion of the calvaria with the external surface (scalp side) placed next to the pH sensor was found to be more responsive to hPTH(1–34) than the internal side (brain side) (Fig. 3, left panel). For calcitonin, a greater response was observed when the internal side of the calvarium was placed next to the pH sensor (Fig. 3, right panel), findings consistent with the known distributions of osteoblasts and osteoclasts.(17) No response was produced by 100 nM bPTH(7–34) (Fig. 4), hPTH(53–84), or 100 nM [Nle8,18, Tyr34]bPTH(3–34)NH2 (data not shown).
Role of the cAMP/PKA signaling pathway in PTH-induced acidification in calvaria
The cAMP analogs dibutyryl cAMP and 8-bromo-cAMP (8br-cAMP) elicited large increases in ECAR in mouse calvaria (Fig. 5), as did the adenylyl cyclase activator forskolin (Figs. 5A and 5B). Pretreatment of bones with 2 mM dibutyryl cAMP or 8br-cAMP, to maintain the cAMP/PKA pathway in an activated state, caused marked decreases in the responses to 100 nM hPTH(1–34) and 10 μM forskolin (Fig. 5). These results are consistent with the hypothesis that the cAMP/PKApathway mediates the ECAR response to PTH in calvaria. To test further this hypothesis, the bones were pretreated with the phosphodiesterase inhibitor IBMX to amplify the cAMP signal.(18) When a submaximal concentration of PTH was used in the presence of 0.1 mM IBMX, the ECAR response to PTH was clearly enhanced (Fig. 6). IBMX also potentiated the response to a submaximal concentration of forskolin (Fig. 6). Taken together, these findings indicate that the ECAR response to PTH in calvaria is transduced, in large measure, via the cAMP/PKA signaling pathway.
Role of the inositol lipid/Ca2+/PKC pathway in PTH-induced ECAR in calvaria
A high concentration of the PKC activator PMA elicited a small increase in ECAR, which persisted for at least 90 minutes after removal of PMA from the perfusing medium (Fig. 7). The response to PTH was unaffected after pretreatment with 1 μM PMA for 60 minutes (Fig. 8), suggesting that the PTH-induced increase in ECAR was not mediated by phorbol ester-regulated PKCs. To test the possible role of changes in [Ca2+]i induced by PTH, intracellular calcium stores were released by treatment of calvaria with 2 μM Tg; a small transient increase in ECAR was observed (Fig. 9), indicating that an increase in [Ca2+]i induced pharmacologically can cause an increase in ECAR. However, pretreatment with 2 μM Tg had no affect on the subsequent response to PTH (Fig. 10). The small increases in ECAR induced by PMA alone and Tg alone and the inability of pretreatment with these agents to blunt or prevent the response to PTH suggest that, in this system, the inositol lipid signaling pathway is poorly coupled to the cellular machinery responsible for increases in extracellular acidification and that this pathway is not the major signaling pathway for PTH-induced ECAR in mouse calvaria.
Role of carbonic anhydrase in PTH-enhanced ECAR in calvaria
To determine if osteoclasts contribute to the PTH-induced acidification via carbonic anhydrase-dependent proton extrusion, the response to PTH was measured in the presence of the carbonic anhydrase inhibitors ethoxolamide (4 μM) or acetazolamide (100 μM). Neither inhibitor affected the PTH-induced ECAR response, whereas they both inhibited PTH-induced calcium release from mouse calvaria in an in vitro bone resorption assay (data not shown). These results indicate that the ECAR response to PTH in intact calvaria is not likely to be mediated by carbonic anhydrase–dependent acid production by osteoclasts.
Because of the relatively low acidification rates associated with intact calvarial tissue, we modified the manner in which data were collected on the Cytosensor. Instead of the usual method of measuring the acidification rate while the pump was off for 15–30 s once per cycle, the pump ran continuously, and we calculated the slope of the voltage change for a ∼150-s interval every 3 minutes, reporting these data as the change in pH versus time, rather than absolute ECAR. Low acidification rates in tissue compared with cells in culture are consistent with reports of active glycolysis in cultured cells while aerobic respiration predominates in tissue.(3) Respiration produces significantly fewer protons per adenosine triphosphate (ATP) generated than glycolysis, leading to difficulties in using the conventional protocol for measuring hormonal responses in intact tissue. Our method of collecting acidification data overcame this limitation and may be useful in assaying ECAR responses in other tissues. Consistent with our previous report in SaOS-2 cells, PTH produced a dose-dependent increase in ECAR in intact bone; therefore, the PTH-induced increase in ECAR observed in human osteosarcoma cells is not an artifact of cultured neoplastic cells.(1)
The 0.11-pH-unit decrease occurring over 1 h of PTH treatment observed in our experiments is large enough to cause measurable changes in osteoclast activity.(16) Neuman et al. found, in animal experiments, that citrate in spongiosal blood from the femur increased in less than 1 h after parathyroid extract injection.(19) Assuming the citrate was excreted as the acid, these results in dogs are consistent with the time course of our findings in mouse bone. We conclude that the effects of PTH on acid extrusion by intact tissue in vitro can be measured using microphysiometry, and this approach may be useful in understanding the response of this tissue to intermittent PTH treatment.
The increase in ECAR elicited by PTH required the first three residues of the 1–34 amino acid fragment of PTH, because [Tyr34]bPTH(7–34)NH2 and [Nle8,18, Tyr34]bPTH(3–34)NH2 were inactive. These results also showed that the ECAR response is not caused by nonspecific effects of the polypeptide on the sensor or tissue and are consistent with the known structure-activity relationships for PTH.(20) Lack of response to hPTH(53–84) is taken as evidence against a carboxy-terminal PTH receptor in this tissue.
Our results are consistent with the ECAR response to PTH as mediated by the cAMP/PKA signal transduction pathway in mouse calvaria. We showed that activation of PKA by forskolin, cAMP analogs, and IBMX caused increases in ECAR. Pretreatment with IBMX increased the response to a submaximal concentration of PTH as expected for a cAMP/PKA-mediated signal. Furthermore, maintaining the cAMP/PKA pathway in an activated state by pretreatment with cAMP analogs, greatly reduced the response to PTH. In contrast to cAMP/PKA pathway activation, stimulation of the inositol lipid/Ca2+/PKC pathway by PMA and Tg elicited only small ECAR responses, and pretreatment with PMA or Tg did not alter the response to PTH.
Because osteoclasts resorb bone by acidifying the resorption pit, we sought to test if osteoclast-mediated acidification was a major component of the PTH-induced increase in ECAR. Carbonic anhydrase inhibitors have been shown to inhibit bone resorption.(21) Ethoxolamide and acetazolamide had no effect on the PTH-induced change in ECAR, but they each inhibited bone resorption in the same calvarial tissue, indicating that carbonic anhydrase-mediated acid extrusion was not the major pathway for the PTH-induced increase in ECAR in this tissue. Thus, the rapid ECAR response to PTH probably is caused by the response in osteoblastic cells, consistent with our previous findings.(1) It is likely that longer experiments would be required to study the response of osteoclasts.
In contrast to previous results in human osteoblast-like SAOS-2 cells, in which the acute ECAR response to PTH was mediated by the inositol lipid/Ca2+/PKC pathway, we found that the cAMP/PKA pathway mediated the response of mouse calvarial tissue. Direct comparisons of the two systems are complicated by the differences in the manner in which the data were collected. In calvaria, the minimum time for the rate measurement was 3 minutes, whereas measurements were obtained with cultured cells in less than 1 minute. Barrett et al. did not study the longer term (>1 minute) ECAR responses of SAOS-2 cells to PTH; therefore, the results on the signal transduction pathway in calvaria are not necessarily inconsistent with the previous report.(1) The lack of a measurable immediate (<1 minute) response in calvarial tissue to PTH may be a technical limitation of using the intact tissue. Alternatively, because the ECAR response to pharmacologic activation of the inositol lipid pathway was small in calvaria, it could have been missed in our experimental protocol. Finally, we cannot rule out slow diffusion rates of PTH into the tissue and of acid metabolites out of the tissue as an explanation of the temporal differences in ECAR response of cells versus intact tissue.
We thank Jean Foley for help in the preparation of this manuscript. This investigation was supported in part by a research grant from the National Institute of Diabetes, Digestive and Kidney Diseases (DK 10206).