0013-7227/90/1264-1941$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 4 Printed in U.S.A.
Stimulation by Parathyroid Hormone of 45Ca2+ Uptake in Osteoblast-Like Cells: Possible Involvement of Alkaline Phosphatase* SHOICHI FUKAYAMAf AND ARMEN H. TASHJIAN, JR. Laboratory of Toxicology, Harvard School of Public Health, and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
and 46Ca2+ uptake. SaOS-2 cells contained high levels of alkaline phosphatase activity and continuously released the enzyme into the medium. Release was enhanced by treatment with hPTH(1-34) for 10 min. Incubation of cells with levamisole (an inhibitor of the liver/bone/kidney type of ALPase) resulted in a rapid decrease in basal and PTH-stimulated 45Ca2+ uptake, while treatment with L-Phe-Gly-Gly (an inhibitor of human placental ALPase) was without effect. Treatment of the cells with ALPase (bovine kidney) enhanced 45Ca2+ uptake. In MG-63 cells, a stimulatory effect of hPTH-(l-34) on cell-associated 46Ca2+ was also observed; however, hPTH-(l-34) did not stimulate cAMP production in MG-63 cells. In ROS 17/2.8 cells, neither hPTH(1-34) nor rat PTH-(l-34) stimulated an increase in cell-associated 45Ca2+, while in UMR-106 cells, rat PTH-(l-34) and (Bu)2cAMP did enhance 45Ca2+ uptake, although hPTH-(l-34) was without effect. We conclude that PTH can stimulate an increase in cell-associated 45Ca2+ in several osteoblast-like cell lines, possibly by modulating local ALPase activity; however, this action of PTH does not appear to be obligatorily dependent on the adenylate cyclase-stimulating action of PTH. (Endocrinology 126: 1941-1949, 1990)
ABSTRACT. We have investigated the actions of human PTH [hPTH-(l-34)] on the association of 45Ca2+ with two human (SaOS-2 and MG-63) and two rat (ROS 17/2.8 and UMR-106) osteoblast-like cell types. In SaOS-2 cells, hPTH-(l-34) binds to specific membrane receptors to activate adenylate cyclase. Treatment of SaOS-2 cells with hPTH-(l-34) resulted in an increase in 45Ca2+ uptake, in a dose-dependent fashion, up to 2to 4-fold above control values. The increase was first evident at 10 min and persisted for at least 30 min. Treatment with nimodipine, a calcium channel antagonist, was without effect on the stimulatory action of PTH. A similar enhancement of cellassociated 45Ca2+ was observed when the cells were incubated with vasoactive intestinal peptide, which acts via different receptors to activate adenylate cyclase in SaOS-2 cells. Treatment with (Bu)2cAMP also induced an increase in cell-associated 46 Ca2+. Pretreatment of SaOS-2 cells with hPTH-(l-34) for 4 h, which induced homologous desensitization to a second challenge with the same peptide for stimulation of cAMP production, did not attenuate the further enhancement of cell-associated 45Ca2+ by a second treatment with hPTH-(l-34). We then examined a possible relationship between alkaline phosphatase (ALPase)
I
T IS well accepted that PTH acts via stimulation of adenylate cyclase to produce cAMP and subsequent phosphorylation of regulatory cellular proteins by protein kinase-A in osteoblastic cells (1-3). However, it has also been shown that PTH can mobilize cellular calcium, which may then act as an intracellular second messenger in bone cells (4-11). The mechanism by which PTH acts to increase cytosolic free calcium ([Ca2+];) involves at least two processes. The first depends on calcium release from intracellular stores. There is increasing evidence that implicates involvement of the hydrolysis of inositol phospholipids to produce inositol 1,4,5-trisphosphate and diacylglycerol in the rapid release of calcium from Received October 2, 1989. Address all correspondence and requests for reprints to: Armen H. Tashjian, Jr., Laboratory of Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115. * This investigation work was supported in part by a research grant from the NIDDK (DK-10206). t Visiting scientist on leave from the Kaken Pharmaceutical Co. (Tokyo, Japan).
intracellular stores in bone cells (6-9) as well as kidney cells (12, 13). In addition, a second process involves calcium influx through a specific plasma membrane channel(s) (7, 8, 14-17). Several in vitro studies have shown that PTH, prostaglandin E2, and cAMP stimulate calcium uptake in osteoblast-like cells (18-20). Although these findings have suggested the involvement of cAMP in the stimulatory action of PTH on calcium uptake, the precise mechanism is still unknown. Although alkaline phosphatase [orthophosphoricmonoester phosphohydrolase, alkaline optimum] (ALPase) appears to be involved in the process of skeletal mineralization (21-26), the complete spectrum of actions of this enzyme in mineral ion homeostasis in osteoblasts remains unclear (27). In the intestine, it has been suggested that ALPase may be involved in calcium transport (28). A role for ALPase in bone cell calcium transport has not been investigated directly. In this paper we demonstrate that PTH, vasoactive
1941
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
1942
PTH-STIMULATED
45
Ca2+ UPTAKE IN OSTEOBLASTS
intestinal peptide (VIP), and cAMP can stimulate calcium uptake in several types of cultured osteoblast-like bone cells and present evidence that this action of PTH is not obligatorily dependent on the adenylate cyclasestimulating effect of the hormone, but may involve the action of ALPase.
Materials and Methods Materials Culture media and sera for cell culture were purchased from Gibco (Grand Island, NY), and tissue culture plasticware was obtained from Becton Dickinson Co. (Oxnard, CA). Synthetic porcine VIP (lots 76F-06321 and 18F-01711), isobutylmethylxanthine (IBMX), the cAMP standard, (Bu)2cAMP (DBcAMP; lots 49F-7270 and 67F-7296), levamisole (lot 47F-3449), L-Phe-Gly-Gly (lot 104F-0233), ALPase (bovine kidney, affinity purified; lots 48F-8030, 116F-81451, 48F-8040, and 48F-8100), Triton X-100, phosphatase substrate, p-nitrophenol, Tris-[hydroxymethyl]aminomethane, and magnesium chloride (MgCl2) were purchased from Sigma Chemical Co. (St. Louis, MO). Calf intestinal alkaline phosphatase (lot 11383221-23) was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Labeled tracer for the RIA of cAMP, [125I]2'-O-succinyl-(iodotyrosine methyl ester)cAMP (SA, 104-1466 mCi/Vmol), was obtained from ICN ImmunoBiologicals (Lisle, IL). Synthetic human (h) PTH-(1-34) (lot 011237), rat PTH-(l-34) (lot 006735), and hPTH-related protein [hPTHrP-(l-34); lot 013785] were obtained from Peninsula Laboratories, Inc. (Belmont, CA). Nimodipine was kindly provided by Dr. A. Scriabine (Miles Institute, New Haven, CT). 45 CaCl2 (2-3 Ci/mmol) and Aquasol were obtained from New England Nuclear (Billerica, MA). Cell culture Two human (SaOS-2 and MG-63) and two rat (UMR-106 and ROS 17/2.8) osteosarcoma cell lines were used in the present study. The clonal line SaOS-2 (29-31) was originally obtained from Dr. E. Lloyd (Argonne National Laboratory) with the permission of Dr. Jorgen Fogh (Sloan-Kettering Memorial Cancer Center, New York, NY). MG-63 cells (32-35) were obtained from Dr. R. T. Franceschi, currently at the University of Texas: UMR-106 cells (36) were originally provided by Dr. T. J. Martin. ROS 17/2.8 cells (37) were kindly given to us by Dr. G. A. Rodan (Merck Sharp and Dohme, West Point, PA). SaOS-2, MG-63, and UMR-106 cells were cultured at 37 C as monolayers in Eagle's Minimum Essential Medium supplemented (vol/vol) with 10% fetal bovine serum (hereafter MEM+) in a humidified atmosphere of 95% air and 5% CO2. ROS 17/2.8 cells were cultured as monolayers in Ham's F10 nutrient mixture supplemented with 15% horse serum and 2.5% fetal bovine serum (hereafter F10+) under the conditions described above. At the end of each experiment cell number was determined by hemocytometer.
Endo • 1990 Vol 126 • No 4
Calcium uptake Calcium uptake was measured using 45CaCl2. Near-confluent cultures were washed three times with serum-free MEM lacking phenol red (SaOS-2 and UMR-106) or with serum-free Ham's F-10 medium (F10"; ROS 17/2.8) and then preincubated for 24-48 h in the appropriate serum-free medium. After preincubation, fresh serum-free medium (pH 7.4) with 45Ca2+ (1.0 fiCi/ ml) without or with test substances was added. Because MG63 cells did not tolerate serum-free conditions, we eliminated the preincubation with these cells; however, they were washed three times with MEM" just before each experiment. Concentrations of total calcium in MEM" and F10" were 1.8 and 0.3 mM, respectively. When cells were tested with nimodipine, all experiments were performed in a darkened room. After the time intervals indicated in Results, cells were washed rapidly three times with cold (4 C) Ca2+-free Hanks' Buffered Salt Solution (54 mM KC1, 116 mM NaCl, 26 mM NaHCO3) 1.0 mM NaH 2 PO 4 H 2 O, and 5.5 mM D-glucose) containing 0.1 mM LaCl3. The cells were then solubilized with 0.1 N NaOH, and the cell-associated radioactivity was measured by liquid scintillation spectrometry in Aquasol. Net 45Ca2+ uptake was calculated by subtracting the zero time value from each uptake measurement (38). Measurement of cAMP The concentration of cAMP secreted into the medium was measured by RIA, as described by us previously (39, 40). ALPase assay Medium or cellular ALPase activities were determined by measuring the release of p-nitrophenol from p-nitrophenylphosphate spectrophotometrically (405 nm) at 37 C. The assay mixture (pH 10.5) contained 2 mM p-nitrophenylphosphate, 200 mM Tris-[hydroxymethyl]aminomethane, 5 mM MgCl2, and 0.2% Triton X-100. The reaction was stopped by adding 0.05 N NaOH. Measurement of protein Protein concentrations were determined by the Bio-Rad method (according to the manufacturer's procedure). Statistical analysis All results were expressed as the mean ± SE. Statistical analyses were made by Student's t test.
Results 45
Actions of PTH on Ca
2+
uptake
In the human osteoblast-like cell line SaOS-2, hPTH(1-34) stimulated the uptake of 45Ca2+ in a dose-dependent fashion by up to 4-fold above the control level (Fig. 1). The increase was first evident at 10 min and persisted for at least 30 min (Table 1). Treatment with nimodipine (100 nM), a voltage-dependent calcium channel blocker
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
PTH-STIMULATED
45
Ca2+ UPTAKE IN OSTEOBLASTS
1943
i 5
I
hPTH
i-34 FIG. 1. Dose-response curve for the enhancement of 45Ca2+ uptake by hPTH-(l-34) in SaOS-2 cells. Cells were incubated in serum-free, phenol red-free medium for 24 h. hPTH-(l-34) was added simultaneously with 4SCa2+, and the uptake was measured at 15 min. Each point gives the mean value, and the brackets give the SE for groups of four or five dishes. TABLE 1. Time course of the actions of hPTH-(l-34) and DBcAMP on 46Ca2+ uptake in SaOS-2 cells Uptake of 45Ca2+ (cpm X 10"4/mg protein)" Time (min) 0 5 10 15 30
Control 0.7 ± 1.4 ± 1.7 ± 2.8 ± 18 ±
0.05 0.13 0.13 0.10 2.1
hPTH-(l-34) (100 ng/ml)
DBcAMP (1 mM)
1.4 ± 0.10 2.2 ± 0.11" 4.7 ± 0.54* 24 ± 0.60"
6.1 ± 0.61* 24 ± 1.5*
Cells were preincubated in serum-free, phenol red-free medium for 48 h. hPTH-(l-34) or DBcAMP was added with 45Ca2+ at zero time. Uptake of 48Ca2+ was determined for the time intervals indicated. 0 Each value gives the mean ± SE for four dishes. * Significant increase above control (P < 0.05) was first observed at 10 min.
(41), did not inhibit the stimulatory effect of PTH on Ca2+ uptake (Fig. 2). On the contrary, 100 nM nimodipine itself significantly stimulated the basal uptake of 45 Ca2+. Enhancement of cell-associated 45Ca2+ was also observed when the cells were treated with VIP (Fig. 2) or DBcAMP (Table 1). A stimulatory effect of hPTH-(l34) on 45Ca2+ uptake was also observed in a dose-dependent fashion in another human osteoblast-like cell line, MG-63 (Fig. 3). The effect of PTH was first evident at 15 min in MG-63 cells (Table 2). PTHrP-(l-34) had an effect similar to that of hPTH-(l-34) (Table 2). However, hPTH-(l-34), hPTHrP-(l-34), and VIP did not stimulate cAMP production in MG-63 cells (Table 3). In rat ROS 17/2.8 cells, hPTH-(l-34), hPTHrP-(l34), and rat PTH-(l-34) did not stimulate an increase 45
I
Control
Nimodipine
FIG. 2. Effects of the calcium channel blocker (nimodipine, 100 nM) on the uptake of 4SCa2+ stimulated by hPTH-(l-34) or VIP in SaOS-2 cells. Cells were incubated in serum-free, phenol red-free medium for 24 h before the addition of stimulators. The cells were then incubated in 45Ca2+-containing medium for 15 min in the absence or presence of nimodipine without or with hPTH-(l-34) (100 ng/ml) or VIP (10~8 M). Each bar gives the mean value, and the brackets give the SE for four dishes. *, P < 0.05; **, P < 0.01 {vs. control value in the same group). t, P < 0.05 (vs. appropriate nonnimodipine-treated control group).
•S
V5
26
22
K ^ 18 14
10
0
0.1
10 (ng/ml)
100
FIG. 3. Dose-response curve for the enhancement of 46Ca2+ uptake by hPTH-(l-34) in MG-63 cells. Cells were grown to confluence and then washed three times with serum-free, phenol red-free medium. hPTH(1-34) was added with 45Ca2+, and the uptake was measured at 15 min. Each point gives the mean value, and the brackets give the SE for groups of four dishes.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
45
Ca2+ UPTAKE IN OSTEOBLASTS
PTH-STIMULATED
1944
TABLE 2. Time course of the actions of hPTH-(l-34) and hPTHrP(1-34) on 45Ca2+ uptake in MG-63 cells Uptake of 45Ca2+ (cpm x 10"4/mg protein)" (min) 0 5 10 15
30
2.8 ± 3.9 ± 3.2 ± 3.5 ± 16 ±
hPTHrP-(l-34) (100 ng/ml)
hPTH-(l-34) (100 ng/ml)
Pnntrnl
0.26 0.70 0.26 0.28 1.30
3.0 ± 3.3 ± 6.3 ± 20 ±
0.20 0.42 0.36* 1.7C
4.3 ± 3.4 ± 7.2 ± 21 ±
0.09 0.27 0.30" 0.396
Cells were grown to confluence and then washed three times with serum-free, phenol red-free medium. Hormones were added with 45Ca2+ at zero time. Uptake of 45Ca2+ was determined for the designated time periods. "Each value gives the mean ± SE for four dishes. Significant increases in 45Ca2+ uptake were first observed at 15 min. "P
Vol. 126, No. 4 Printed in U.S.A.
Stimulation by Parathyroid Hormone of 45Ca2+ Uptake in Osteoblast-Like Cells: Possible Involvement of Alkaline Phosphatase* SHOICHI FUKAYAMAf AND ARMEN H. TASHJIAN, JR. Laboratory of Toxicology, Harvard School of Public Health, and the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
and 46Ca2+ uptake. SaOS-2 cells contained high levels of alkaline phosphatase activity and continuously released the enzyme into the medium. Release was enhanced by treatment with hPTH(1-34) for 10 min. Incubation of cells with levamisole (an inhibitor of the liver/bone/kidney type of ALPase) resulted in a rapid decrease in basal and PTH-stimulated 45Ca2+ uptake, while treatment with L-Phe-Gly-Gly (an inhibitor of human placental ALPase) was without effect. Treatment of the cells with ALPase (bovine kidney) enhanced 45Ca2+ uptake. In MG-63 cells, a stimulatory effect of hPTH-(l-34) on cell-associated 46Ca2+ was also observed; however, hPTH-(l-34) did not stimulate cAMP production in MG-63 cells. In ROS 17/2.8 cells, neither hPTH(1-34) nor rat PTH-(l-34) stimulated an increase in cell-associated 45Ca2+, while in UMR-106 cells, rat PTH-(l-34) and (Bu)2cAMP did enhance 45Ca2+ uptake, although hPTH-(l-34) was without effect. We conclude that PTH can stimulate an increase in cell-associated 45Ca2+ in several osteoblast-like cell lines, possibly by modulating local ALPase activity; however, this action of PTH does not appear to be obligatorily dependent on the adenylate cyclase-stimulating action of PTH. (Endocrinology 126: 1941-1949, 1990)
ABSTRACT. We have investigated the actions of human PTH [hPTH-(l-34)] on the association of 45Ca2+ with two human (SaOS-2 and MG-63) and two rat (ROS 17/2.8 and UMR-106) osteoblast-like cell types. In SaOS-2 cells, hPTH-(l-34) binds to specific membrane receptors to activate adenylate cyclase. Treatment of SaOS-2 cells with hPTH-(l-34) resulted in an increase in 45Ca2+ uptake, in a dose-dependent fashion, up to 2to 4-fold above control values. The increase was first evident at 10 min and persisted for at least 30 min. Treatment with nimodipine, a calcium channel antagonist, was without effect on the stimulatory action of PTH. A similar enhancement of cellassociated 45Ca2+ was observed when the cells were incubated with vasoactive intestinal peptide, which acts via different receptors to activate adenylate cyclase in SaOS-2 cells. Treatment with (Bu)2cAMP also induced an increase in cell-associated 46 Ca2+. Pretreatment of SaOS-2 cells with hPTH-(l-34) for 4 h, which induced homologous desensitization to a second challenge with the same peptide for stimulation of cAMP production, did not attenuate the further enhancement of cell-associated 45Ca2+ by a second treatment with hPTH-(l-34). We then examined a possible relationship between alkaline phosphatase (ALPase)
I
T IS well accepted that PTH acts via stimulation of adenylate cyclase to produce cAMP and subsequent phosphorylation of regulatory cellular proteins by protein kinase-A in osteoblastic cells (1-3). However, it has also been shown that PTH can mobilize cellular calcium, which may then act as an intracellular second messenger in bone cells (4-11). The mechanism by which PTH acts to increase cytosolic free calcium ([Ca2+];) involves at least two processes. The first depends on calcium release from intracellular stores. There is increasing evidence that implicates involvement of the hydrolysis of inositol phospholipids to produce inositol 1,4,5-trisphosphate and diacylglycerol in the rapid release of calcium from Received October 2, 1989. Address all correspondence and requests for reprints to: Armen H. Tashjian, Jr., Laboratory of Toxicology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115. * This investigation work was supported in part by a research grant from the NIDDK (DK-10206). t Visiting scientist on leave from the Kaken Pharmaceutical Co. (Tokyo, Japan).
intracellular stores in bone cells (6-9) as well as kidney cells (12, 13). In addition, a second process involves calcium influx through a specific plasma membrane channel(s) (7, 8, 14-17). Several in vitro studies have shown that PTH, prostaglandin E2, and cAMP stimulate calcium uptake in osteoblast-like cells (18-20). Although these findings have suggested the involvement of cAMP in the stimulatory action of PTH on calcium uptake, the precise mechanism is still unknown. Although alkaline phosphatase [orthophosphoricmonoester phosphohydrolase, alkaline optimum] (ALPase) appears to be involved in the process of skeletal mineralization (21-26), the complete spectrum of actions of this enzyme in mineral ion homeostasis in osteoblasts remains unclear (27). In the intestine, it has been suggested that ALPase may be involved in calcium transport (28). A role for ALPase in bone cell calcium transport has not been investigated directly. In this paper we demonstrate that PTH, vasoactive
1941
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
1942
PTH-STIMULATED
45
Ca2+ UPTAKE IN OSTEOBLASTS
intestinal peptide (VIP), and cAMP can stimulate calcium uptake in several types of cultured osteoblast-like bone cells and present evidence that this action of PTH is not obligatorily dependent on the adenylate cyclasestimulating effect of the hormone, but may involve the action of ALPase.
Materials and Methods Materials Culture media and sera for cell culture were purchased from Gibco (Grand Island, NY), and tissue culture plasticware was obtained from Becton Dickinson Co. (Oxnard, CA). Synthetic porcine VIP (lots 76F-06321 and 18F-01711), isobutylmethylxanthine (IBMX), the cAMP standard, (Bu)2cAMP (DBcAMP; lots 49F-7270 and 67F-7296), levamisole (lot 47F-3449), L-Phe-Gly-Gly (lot 104F-0233), ALPase (bovine kidney, affinity purified; lots 48F-8030, 116F-81451, 48F-8040, and 48F-8100), Triton X-100, phosphatase substrate, p-nitrophenol, Tris-[hydroxymethyl]aminomethane, and magnesium chloride (MgCl2) were purchased from Sigma Chemical Co. (St. Louis, MO). Calf intestinal alkaline phosphatase (lot 11383221-23) was purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Labeled tracer for the RIA of cAMP, [125I]2'-O-succinyl-(iodotyrosine methyl ester)cAMP (SA, 104-1466 mCi/Vmol), was obtained from ICN ImmunoBiologicals (Lisle, IL). Synthetic human (h) PTH-(1-34) (lot 011237), rat PTH-(l-34) (lot 006735), and hPTH-related protein [hPTHrP-(l-34); lot 013785] were obtained from Peninsula Laboratories, Inc. (Belmont, CA). Nimodipine was kindly provided by Dr. A. Scriabine (Miles Institute, New Haven, CT). 45 CaCl2 (2-3 Ci/mmol) and Aquasol were obtained from New England Nuclear (Billerica, MA). Cell culture Two human (SaOS-2 and MG-63) and two rat (UMR-106 and ROS 17/2.8) osteosarcoma cell lines were used in the present study. The clonal line SaOS-2 (29-31) was originally obtained from Dr. E. Lloyd (Argonne National Laboratory) with the permission of Dr. Jorgen Fogh (Sloan-Kettering Memorial Cancer Center, New York, NY). MG-63 cells (32-35) were obtained from Dr. R. T. Franceschi, currently at the University of Texas: UMR-106 cells (36) were originally provided by Dr. T. J. Martin. ROS 17/2.8 cells (37) were kindly given to us by Dr. G. A. Rodan (Merck Sharp and Dohme, West Point, PA). SaOS-2, MG-63, and UMR-106 cells were cultured at 37 C as monolayers in Eagle's Minimum Essential Medium supplemented (vol/vol) with 10% fetal bovine serum (hereafter MEM+) in a humidified atmosphere of 95% air and 5% CO2. ROS 17/2.8 cells were cultured as monolayers in Ham's F10 nutrient mixture supplemented with 15% horse serum and 2.5% fetal bovine serum (hereafter F10+) under the conditions described above. At the end of each experiment cell number was determined by hemocytometer.
Endo • 1990 Vol 126 • No 4
Calcium uptake Calcium uptake was measured using 45CaCl2. Near-confluent cultures were washed three times with serum-free MEM lacking phenol red (SaOS-2 and UMR-106) or with serum-free Ham's F-10 medium (F10"; ROS 17/2.8) and then preincubated for 24-48 h in the appropriate serum-free medium. After preincubation, fresh serum-free medium (pH 7.4) with 45Ca2+ (1.0 fiCi/ ml) without or with test substances was added. Because MG63 cells did not tolerate serum-free conditions, we eliminated the preincubation with these cells; however, they were washed three times with MEM" just before each experiment. Concentrations of total calcium in MEM" and F10" were 1.8 and 0.3 mM, respectively. When cells were tested with nimodipine, all experiments were performed in a darkened room. After the time intervals indicated in Results, cells were washed rapidly three times with cold (4 C) Ca2+-free Hanks' Buffered Salt Solution (54 mM KC1, 116 mM NaCl, 26 mM NaHCO3) 1.0 mM NaH 2 PO 4 H 2 O, and 5.5 mM D-glucose) containing 0.1 mM LaCl3. The cells were then solubilized with 0.1 N NaOH, and the cell-associated radioactivity was measured by liquid scintillation spectrometry in Aquasol. Net 45Ca2+ uptake was calculated by subtracting the zero time value from each uptake measurement (38). Measurement of cAMP The concentration of cAMP secreted into the medium was measured by RIA, as described by us previously (39, 40). ALPase assay Medium or cellular ALPase activities were determined by measuring the release of p-nitrophenol from p-nitrophenylphosphate spectrophotometrically (405 nm) at 37 C. The assay mixture (pH 10.5) contained 2 mM p-nitrophenylphosphate, 200 mM Tris-[hydroxymethyl]aminomethane, 5 mM MgCl2, and 0.2% Triton X-100. The reaction was stopped by adding 0.05 N NaOH. Measurement of protein Protein concentrations were determined by the Bio-Rad method (according to the manufacturer's procedure). Statistical analysis All results were expressed as the mean ± SE. Statistical analyses were made by Student's t test.
Results 45
Actions of PTH on Ca
2+
uptake
In the human osteoblast-like cell line SaOS-2, hPTH(1-34) stimulated the uptake of 45Ca2+ in a dose-dependent fashion by up to 4-fold above the control level (Fig. 1). The increase was first evident at 10 min and persisted for at least 30 min (Table 1). Treatment with nimodipine (100 nM), a voltage-dependent calcium channel blocker
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
PTH-STIMULATED
45
Ca2+ UPTAKE IN OSTEOBLASTS
1943
i 5
I
hPTH
i-34 FIG. 1. Dose-response curve for the enhancement of 45Ca2+ uptake by hPTH-(l-34) in SaOS-2 cells. Cells were incubated in serum-free, phenol red-free medium for 24 h. hPTH-(l-34) was added simultaneously with 4SCa2+, and the uptake was measured at 15 min. Each point gives the mean value, and the brackets give the SE for groups of four or five dishes. TABLE 1. Time course of the actions of hPTH-(l-34) and DBcAMP on 46Ca2+ uptake in SaOS-2 cells Uptake of 45Ca2+ (cpm X 10"4/mg protein)" Time (min) 0 5 10 15 30
Control 0.7 ± 1.4 ± 1.7 ± 2.8 ± 18 ±
0.05 0.13 0.13 0.10 2.1
hPTH-(l-34) (100 ng/ml)
DBcAMP (1 mM)
1.4 ± 0.10 2.2 ± 0.11" 4.7 ± 0.54* 24 ± 0.60"
6.1 ± 0.61* 24 ± 1.5*
Cells were preincubated in serum-free, phenol red-free medium for 48 h. hPTH-(l-34) or DBcAMP was added with 45Ca2+ at zero time. Uptake of 48Ca2+ was determined for the time intervals indicated. 0 Each value gives the mean ± SE for four dishes. * Significant increase above control (P < 0.05) was first observed at 10 min.
(41), did not inhibit the stimulatory effect of PTH on Ca2+ uptake (Fig. 2). On the contrary, 100 nM nimodipine itself significantly stimulated the basal uptake of 45 Ca2+. Enhancement of cell-associated 45Ca2+ was also observed when the cells were treated with VIP (Fig. 2) or DBcAMP (Table 1). A stimulatory effect of hPTH-(l34) on 45Ca2+ uptake was also observed in a dose-dependent fashion in another human osteoblast-like cell line, MG-63 (Fig. 3). The effect of PTH was first evident at 15 min in MG-63 cells (Table 2). PTHrP-(l-34) had an effect similar to that of hPTH-(l-34) (Table 2). However, hPTH-(l-34), hPTHrP-(l-34), and VIP did not stimulate cAMP production in MG-63 cells (Table 3). In rat ROS 17/2.8 cells, hPTH-(l-34), hPTHrP-(l34), and rat PTH-(l-34) did not stimulate an increase 45
I
Control
Nimodipine
FIG. 2. Effects of the calcium channel blocker (nimodipine, 100 nM) on the uptake of 4SCa2+ stimulated by hPTH-(l-34) or VIP in SaOS-2 cells. Cells were incubated in serum-free, phenol red-free medium for 24 h before the addition of stimulators. The cells were then incubated in 45Ca2+-containing medium for 15 min in the absence or presence of nimodipine without or with hPTH-(l-34) (100 ng/ml) or VIP (10~8 M). Each bar gives the mean value, and the brackets give the SE for four dishes. *, P < 0.05; **, P < 0.01 {vs. control value in the same group). t, P < 0.05 (vs. appropriate nonnimodipine-treated control group).
•S
V5
26
22
K ^ 18 14
10
0
0.1
10 (ng/ml)
100
FIG. 3. Dose-response curve for the enhancement of 46Ca2+ uptake by hPTH-(l-34) in MG-63 cells. Cells were grown to confluence and then washed three times with serum-free, phenol red-free medium. hPTH(1-34) was added with 45Ca2+, and the uptake was measured at 15 min. Each point gives the mean value, and the brackets give the SE for groups of four dishes.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
45
Ca2+ UPTAKE IN OSTEOBLASTS
PTH-STIMULATED
1944
TABLE 2. Time course of the actions of hPTH-(l-34) and hPTHrP(1-34) on 45Ca2+ uptake in MG-63 cells Uptake of 45Ca2+ (cpm x 10"4/mg protein)" (min) 0 5 10 15
30
2.8 ± 3.9 ± 3.2 ± 3.5 ± 16 ±
hPTHrP-(l-34) (100 ng/ml)
hPTH-(l-34) (100 ng/ml)
Pnntrnl
0.26 0.70 0.26 0.28 1.30
3.0 ± 3.3 ± 6.3 ± 20 ±
0.20 0.42 0.36* 1.7C
4.3 ± 3.4 ± 7.2 ± 21 ±
0.09 0.27 0.30" 0.396
Cells were grown to confluence and then washed three times with serum-free, phenol red-free medium. Hormones were added with 45Ca2+ at zero time. Uptake of 45Ca2+ was determined for the designated time periods. "Each value gives the mean ± SE for four dishes. Significant increases in 45Ca2+ uptake were first observed at 15 min. "P
