Vitamin D-regulated calcium transport unique in vitro model
in Caco-2 cells:
ANNA R. GIULIANO AND RICHARD J. WOOD United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Mineral Bioavailability Laboratory, Boston, Massachusetts 02111
GIULIANO, ANNA R., AND RICHARD J. WOOD. Vitamin Dregulated calcium transport in Caco-2 cells: unique in vitro model. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G207-G212,1991.-The human colon adenocarcinoma cell line Caco-2 is the only intestinal cell line to differentiate spontaneously in culture exhibiting structural and biochemical characteristics of mature enterocytes and to possess a vitamin D receptor in the fully differentiated state. Transepithelial calcium transport was characterized in differentiated Caco-2 cells grown on permeable filters supports to assess the potential utility of this cell line as an in vitro model to study l,25dihydroxyvitamin D3 [ 1,25(OH)zD3] -induced calcium transport. Calcium transport was increased in a dose-dependent manner by 1,25(OH)2D3. Total calcium transport at different calcium concentrations could be fitted to a modified MichaelisMenten equation containing a linear transport component. The maximum rate of saturable calcium transport was increased by 4.3-fold (P < 0.005) in cells treated with lo-” M 1,25(OH)2DS. This treatment also increased the apparent buffer calcium concentration that results in half-maximal velocity from 0.4 to 1.3 mM but had no significant effect on nonsaturable calcium transport. Caco-2 cells grown on permeable filter supports provide a unique in vitro human cell culture model to study the mechanism of vitamin D-regulated transepithelial intestinal calcium transport.
transepithelial calcium transport; intestine; 1,25-dihydroxycholecalciferol; cultured human intestinal cells I KINETICS OF IN VIVO intestinal calcium absorption can be described as the sum of two processes: 1) a saturable process, presumably representing carrier-mediated, transcellular transport of calcium, and 2) a nonsaturable process that may reflect diffusional or paracellular transport (2, 3, 6, 20). In vivo, the maximal rate of calcium transport by the saturable process ( Vmax) is influenced by vitamin D status, dietary calcium intake, and age, whereas the nonsaturable process is determined mainly by the luminal calcium concentration (3, 8, 25, 26) Transcellular calcium transport can be divided into three phases: 1) entry at the brush-border membrane, 2) translocation through the cell, and 3) extrusion at the basolateral membrane (3). Various studies have suggested that vitamin D status can affect the rate of calcium transport at each of these three cellular loci. Entry across the brush-border membrane of the enterocyte has been shown to be influenced by vitamin D status (16, 19). The intracellular transport of calcium may also be THE
0193~1857/91
$1.50
Copyright
0 1991
enhanced by 1,25dihydroxyvitamin D, (1,25(OH)zD3), presumably via increases in the cytosolic concentration of calcium-binding protein (3, 18, 29, 30) or uptake of calcium into intracellular organelles, such as Golgi (9, 17) or lysosomal vesicles (21-23). At the basolateral membrane, calcium efflux is presumed to be dependent on Ca’+-Mg2+ adenosinetriphosphatase activity that has been reported to be influenced by vitamin D status (3, 30). Although each of these 1,25(OH)zD3-dependent processes could influence transcellular calcium transport, the molecular details of calcium transport are still not well understood. Progress in the development of an integrated understanding of 1,25(0H)2D3-regulated transcellular calcium transport could be advanced by the availability of a cultured intestinal cell line that could be shown to possessa vitamin D receptor and to demonstrate vitamin D-dependent vectorial calcium transport. The human colon adenocarcinoma cell line Caco-2 has unique properties in that it is the only intestinal cell line that differentiates spontaneously in culture (4), exhibiting structural and functional differentiation patterns characteristic of mature small intestinal enterocytes (27). The differentiated Caco-2 is covered by typical brushborder microvilli and, when cultured in monolayer, possessestight junctions and forms many domes, characteristic of functionally polarized, transporting epithelial cells (12). Ussing chamber studies of Caco-2 cells grown on permeable supports have demonstrated a positive short-circuit current, indicative of net cation absorption or anion secretion (11). Moreover, the functional differentiation of the Caco-2 cell is a growth-related phenomenon (27). The activity of sucrase-isomaltase, alkaline phosphatase, and aminopeptidase, markers of enterocytic differentiation, is low during the log growth phase in these cells but increases markedly after the stationary growth phase (27). In previous studies, we have demonstrated that the Caco-2 cell line possessesa vitamin D receptor similar to that present in mammalian intestine (10). The concentration of vitamin D receptors in Caco-2 cells increases with differentiation paralleling the spontaneous increase in alkaline phosphatase activity. The objective of this study was to determine whether 1,25(OH)pD3 affected transepithelial calcium transport in Caco-2 cells. METHODS
Materials. Crystalline 1,25(OH)zD3 was a gift from Dr. M. Uskokovic of Hoffman LaRoche (Nutley, NJ) and the American
Physiological
Society
G207
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 9, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
G208
VITAMIN
D-REGULATED
CALCIUM
25hydroxyvitamin D, [25(OH)D,] was purchased from Biomol (Plymouth Meeting, PA). Fetal bovine serum (FBS) used for culturing cells was purchased from Hyclone (Logan, UT). Culture media ingredients were purchased from GIBCO (Grand Island, NY). [““Cal and [“Hlinulin (mol wt, 5,200) were purchased from New England Nuclear (Boston, MA). Cell culture. The human colon adenocarcinoma cell line Caco-2 was obtained from the American Type Tissue Collection (passage 21) and studied between the 25th and 50th passages. Cells were routinely grown in 75 cm2 plastic flasks (Corning) in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose supplemented with 1% nonessential amino acids, 20% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mg/ml gentamicin sulfate. Caco-2 cells were maintained at 37°C in a 5% CO,95% air atmosphere. Cells were seeded at a density of 27 X 10”/cm2 and were routinely passaged at a ratio of 1:4 when 70-80% confluent. Cells used in transepithelial calcium transport experiments were seeded at 3 x lo’/ insert into Transwell permeable supports (Costar, Cambridge, MA, catalog no. 3412) as per supplier instructions. Media were changed every other day before confluence and every day after confluence. Transepithelial calcium transport studies. Caco-2 cells seeded on permeable supports were grown in culture media containing 20% FBS until differentiated at day 15 in culture. On the day of an experiment, cell monolayers were rinsed twice with Dulbecco’s phosphate-buffered saline that contained (in mM) 138 NaCl, 8 Na2HP04, 2.7 KCl, and 1.5 KH2P04, then the permeable support inserts containing cells were transferred to fresh six-well cluster dishes (Falcon) containing 2.6 ml label-free transport buffer that contained (in mM) 140 NaCl, 5.8 KCl, 0.34 Na2HP04, 0.44 KH2P04, 0.8 MgSO,, 20 N-Z-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 4 glutamine, and 25 glucose, pH 7.4. At time 0, radiolabeled (5 &i [45Ca]/ml or 50 &i [“H]inulin/ml) transport buffer (1.5 ml) was added to the top compartment of the insert and the cluster dishes were incubated at 37°C in a shaking water bath. The calcium concentration in the transport buffer was 0.75-15 mM in experiments determining the kinetics of calcium transport and 7.5 mM in experiments determining the effect of 1,25(OH)2D3. At various times after the addition of the labeled transport buffer, 50 ~1 were sampled from the bottom compartment. In experiments testing the effect of 1,25(OH)2D3 on calcium transport, cells were grown in the normal growth media containing 20% FBS until day 11. Cells were then grown in media containing 5% FBS with either lo-’ M 1,25(OH)2D3 or ethanol vehicle not exceeding 0.1%. 1,25(OH)2D3 and ethanol-containing media were added to both the top and bottom compartments of the cluster dishes. Media were replaced daily for a total of 4 days. Calcium transport experiments were conducted after 1, 2, 3, and 4 days of 1,25(OH)2D3 and control treatment. 1,25(OH)2D3 concentration of culture media containing 5% FBS was -5 x lo-l2 M. Measurements. Radioactivity was determined by liquid scintillation counting using a Packard ZOOOCA liquid scintillation counter. Calcium transport was expressed
TRANSPORT
IN
CACO-2
CELLS
as nanomoles calcium transported to the serosal compartment per minute per well during the 30- to 60-min time point, unless specified otherwise. Kinetic constants for transport experiments were calculated from a computer-fitted Michaelis-Menten equation modified to include a linear diffusional component. The data at each calcium concentration were weighted by the reciprocal of their variance. The kinetics of calcium transport were determined by fitting the transport rate at various calcium concentrations to the following equation total transport
=
WI + Knax[~l/Kn + [Sl
where C is the nonsaturable linear component, S is the concentration of calcium in the transport buffer, Vmax is the maximal rate of calcium transport of the saturable component, and K, is the buffer calcium concentration that results in half-maximal velocity. Statistics. The statistical significance of treatment effects of calcium transport and calcium kinetic parameters were made using a t test. Data are presented as means t SE. RESULTS
Permeability of Caco-2 cell monolayer. The transport of [3H]inulin, a molecular probe that does not permeate membranes, was assessed in the presence and absence of a cell monolayer to test the permeability of the Caco-2 monolayer grown for 15 days on permeable supports. We found the Caco-2 cell monolayer at day 15 in culture to be essentially impermeable to inulin. Only 0.6% inulin crossed the cell monolayer in a 2-h period. In contrast, 35% diffused across the permeable filters in the absence of a cell layer in the same time period. Kinetics of transepithelial calcium transport. In our initial studies, the rate of transepithelial calcium transport by Caco-2 cells was determined in confluent, differentiated cells grown in media containing 20% FBS until day 15 in culture. We found the transport of calcium into the bottom compartment to be linear up to at least 120 min at calcium concentrations between 0.75 and 15 mM (Fig. 1A). Because transport was linear over this time period, we have estimated the linear transport rate in subsequent experiments using only the 30- and 60-min time points. The concentration dependence of transepithelial calcium transport in Caco-2 cells was determined and found to be nonlinear (Fig. IB), suggesting the presence of a saturable transport component. The data for calcium transport (nmol Ca min-l . well-l) were fitted to the modified Michaelis-Menten equation to estimate the kinetic parameters for the saturable and nonsaturable transport components. The results of three control kinetic experiments conducted in Caco-2 cells that had been grown in culture media supplemented with 20% FBS until day 15 in culture yielded an apparent K, for the saturable transport component of 2.9 t 1 mM, a maximum value for saturable transport ( Vmax)of 1.4 t 0.1 nmol min. well-‘, and a linear nonsaturable transport component (C) of 0.13 t 0.2 nmol~min-l~well-l~mM-l. Effect of 1,25(OH)2D3 on transepithelial calcium transDart. The effect of 1,25(OH)2D3 on calcium transport in l
l
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 9, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
VITAMIN
D-REGULATED
CALCIUM
TRANSPORT
IN
CACO-2
8
hinl
G209
CELLS
4
0
Ca Concentration
(ml41
1. Time course and concentration dependence of calcium transport across monolayers of Caco-2 grown on permeable filter supports for 15 days in culture media containing 20% fetal bovine serum (FBS). A: calcium transport was linear between 5 and 120 min at all calcium concentrations tested between 0.75 and 15 mM. Transport data for 15 mM calcium from a representative experiment are shown for illustration. B: rate of total calcium transport (circle) was estimated from amount of calcium transported to lower compartment between 30 and 60 min after adding 45Ca to top compartment of filter support and plotted against buffer calcium concentration. Data were fitted to modified Michaelis-Menten equation to derive parameters for saturable and nonsaturable calcium transport. n , Saturable calcium transport calculated from this equation; A, nonsaturable transport. Each experimental point represents mean of 3 wells. Data are from 1 representative experiment. Two other experiments were conducted and yielded similar results. In this experiment buffer-calcium concentration that results in half-maximal velocity was 2.9 mM, maximal and nonsaturable linear component rate of calcium transport of saturable components was 1.2 nmol min-’ . well-‘, was 0.16 nmol . min. well-’ mM? FIG.
l
Caco-2 cells was determined in cells grown in media containing 5% FBS from day 11 to 15 in culture in the presence or absence of 10m8 M 1,25(OH)2D3. Calcium transport measured at 7.5 mM calcium in the transport buffer was significantly greater (2.8 t 0.2 vs. 1.2 t 0.1 nmol. min-’ . well-‘, P < 0.001, n = 6 experiments) in cells treated . . with 1,25(OH)zD3 compared with vehicletreated cultures. Effect of 1,25(OH)2D3 concentration on calcium transport. The effect of 1,25(OH)zDs concentration on calcium transport in Caco-2 cells was assessed by incubating cultures for 4 days in media containing 5% FBS in the absence or presence of 10-l’ to 10m7 M 1,25(OH)2D3. The results are illustrated in Fig. 2. With increasing concentrations of 1,25(OH)zD3 there was an apparent dosedependent increase in calcium transport rates. 1,25(OH)2D3 increased calcium transport at 10-l’ M, the lowest concentration tested. In contrast, 10B7 M 25(OH)D3 did not significantly increase calcium transport (data not shown). Time course of 1,25(OH)2D3-induced calcium transport.
Caco-2 cells were cultured until day 11 in media containing 20% FBS. On day 11 the FBS concentration was reduced to 5% and cultures received either IO-’ M 1,25(OH)2D3 or ethanol vehicle for the next 4 days. Calcium transport was measured at 7.5 mM calcium in
the transport buffer after 1,2,3, and 4 days of treatment. Calcium transport was significantly increased by 1,25(OH)zDa after 1 day of treatment (1.9 vs. 2.9 nmol min-’ .well-1, P < 0.005). Longer periods of treatment did not result in further augmentation of the rate of calcium transport in 1,25(0H)zDs-treated cells. An additional study was carried out to investigate whether incubation of cells with 1,25(OH)zD3 for
in Caco-2 cells:
ANNA R. GIULIANO AND RICHARD J. WOOD United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Mineral Bioavailability Laboratory, Boston, Massachusetts 02111
GIULIANO, ANNA R., AND RICHARD J. WOOD. Vitamin Dregulated calcium transport in Caco-2 cells: unique in vitro model. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G207-G212,1991.-The human colon adenocarcinoma cell line Caco-2 is the only intestinal cell line to differentiate spontaneously in culture exhibiting structural and biochemical characteristics of mature enterocytes and to possess a vitamin D receptor in the fully differentiated state. Transepithelial calcium transport was characterized in differentiated Caco-2 cells grown on permeable filters supports to assess the potential utility of this cell line as an in vitro model to study l,25dihydroxyvitamin D3 [ 1,25(OH)zD3] -induced calcium transport. Calcium transport was increased in a dose-dependent manner by 1,25(OH)2D3. Total calcium transport at different calcium concentrations could be fitted to a modified MichaelisMenten equation containing a linear transport component. The maximum rate of saturable calcium transport was increased by 4.3-fold (P < 0.005) in cells treated with lo-” M 1,25(OH)2DS. This treatment also increased the apparent buffer calcium concentration that results in half-maximal velocity from 0.4 to 1.3 mM but had no significant effect on nonsaturable calcium transport. Caco-2 cells grown on permeable filter supports provide a unique in vitro human cell culture model to study the mechanism of vitamin D-regulated transepithelial intestinal calcium transport.
transepithelial calcium transport; intestine; 1,25-dihydroxycholecalciferol; cultured human intestinal cells I KINETICS OF IN VIVO intestinal calcium absorption can be described as the sum of two processes: 1) a saturable process, presumably representing carrier-mediated, transcellular transport of calcium, and 2) a nonsaturable process that may reflect diffusional or paracellular transport (2, 3, 6, 20). In vivo, the maximal rate of calcium transport by the saturable process ( Vmax) is influenced by vitamin D status, dietary calcium intake, and age, whereas the nonsaturable process is determined mainly by the luminal calcium concentration (3, 8, 25, 26) Transcellular calcium transport can be divided into three phases: 1) entry at the brush-border membrane, 2) translocation through the cell, and 3) extrusion at the basolateral membrane (3). Various studies have suggested that vitamin D status can affect the rate of calcium transport at each of these three cellular loci. Entry across the brush-border membrane of the enterocyte has been shown to be influenced by vitamin D status (16, 19). The intracellular transport of calcium may also be THE
0193~1857/91
$1.50
Copyright
0 1991
enhanced by 1,25dihydroxyvitamin D, (1,25(OH)zD3), presumably via increases in the cytosolic concentration of calcium-binding protein (3, 18, 29, 30) or uptake of calcium into intracellular organelles, such as Golgi (9, 17) or lysosomal vesicles (21-23). At the basolateral membrane, calcium efflux is presumed to be dependent on Ca’+-Mg2+ adenosinetriphosphatase activity that has been reported to be influenced by vitamin D status (3, 30). Although each of these 1,25(OH)zD3-dependent processes could influence transcellular calcium transport, the molecular details of calcium transport are still not well understood. Progress in the development of an integrated understanding of 1,25(0H)2D3-regulated transcellular calcium transport could be advanced by the availability of a cultured intestinal cell line that could be shown to possessa vitamin D receptor and to demonstrate vitamin D-dependent vectorial calcium transport. The human colon adenocarcinoma cell line Caco-2 has unique properties in that it is the only intestinal cell line that differentiates spontaneously in culture (4), exhibiting structural and functional differentiation patterns characteristic of mature small intestinal enterocytes (27). The differentiated Caco-2 is covered by typical brushborder microvilli and, when cultured in monolayer, possessestight junctions and forms many domes, characteristic of functionally polarized, transporting epithelial cells (12). Ussing chamber studies of Caco-2 cells grown on permeable supports have demonstrated a positive short-circuit current, indicative of net cation absorption or anion secretion (11). Moreover, the functional differentiation of the Caco-2 cell is a growth-related phenomenon (27). The activity of sucrase-isomaltase, alkaline phosphatase, and aminopeptidase, markers of enterocytic differentiation, is low during the log growth phase in these cells but increases markedly after the stationary growth phase (27). In previous studies, we have demonstrated that the Caco-2 cell line possessesa vitamin D receptor similar to that present in mammalian intestine (10). The concentration of vitamin D receptors in Caco-2 cells increases with differentiation paralleling the spontaneous increase in alkaline phosphatase activity. The objective of this study was to determine whether 1,25(OH)pD3 affected transepithelial calcium transport in Caco-2 cells. METHODS
Materials. Crystalline 1,25(OH)zD3 was a gift from Dr. M. Uskokovic of Hoffman LaRoche (Nutley, NJ) and the American
Physiological
Society
G207
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 9, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
G208
VITAMIN
D-REGULATED
CALCIUM
25hydroxyvitamin D, [25(OH)D,] was purchased from Biomol (Plymouth Meeting, PA). Fetal bovine serum (FBS) used for culturing cells was purchased from Hyclone (Logan, UT). Culture media ingredients were purchased from GIBCO (Grand Island, NY). [““Cal and [“Hlinulin (mol wt, 5,200) were purchased from New England Nuclear (Boston, MA). Cell culture. The human colon adenocarcinoma cell line Caco-2 was obtained from the American Type Tissue Collection (passage 21) and studied between the 25th and 50th passages. Cells were routinely grown in 75 cm2 plastic flasks (Corning) in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose supplemented with 1% nonessential amino acids, 20% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mg/ml gentamicin sulfate. Caco-2 cells were maintained at 37°C in a 5% CO,95% air atmosphere. Cells were seeded at a density of 27 X 10”/cm2 and were routinely passaged at a ratio of 1:4 when 70-80% confluent. Cells used in transepithelial calcium transport experiments were seeded at 3 x lo’/ insert into Transwell permeable supports (Costar, Cambridge, MA, catalog no. 3412) as per supplier instructions. Media were changed every other day before confluence and every day after confluence. Transepithelial calcium transport studies. Caco-2 cells seeded on permeable supports were grown in culture media containing 20% FBS until differentiated at day 15 in culture. On the day of an experiment, cell monolayers were rinsed twice with Dulbecco’s phosphate-buffered saline that contained (in mM) 138 NaCl, 8 Na2HP04, 2.7 KCl, and 1.5 KH2P04, then the permeable support inserts containing cells were transferred to fresh six-well cluster dishes (Falcon) containing 2.6 ml label-free transport buffer that contained (in mM) 140 NaCl, 5.8 KCl, 0.34 Na2HP04, 0.44 KH2P04, 0.8 MgSO,, 20 N-Z-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 4 glutamine, and 25 glucose, pH 7.4. At time 0, radiolabeled (5 &i [45Ca]/ml or 50 &i [“H]inulin/ml) transport buffer (1.5 ml) was added to the top compartment of the insert and the cluster dishes were incubated at 37°C in a shaking water bath. The calcium concentration in the transport buffer was 0.75-15 mM in experiments determining the kinetics of calcium transport and 7.5 mM in experiments determining the effect of 1,25(OH)2D3. At various times after the addition of the labeled transport buffer, 50 ~1 were sampled from the bottom compartment. In experiments testing the effect of 1,25(OH)2D3 on calcium transport, cells were grown in the normal growth media containing 20% FBS until day 11. Cells were then grown in media containing 5% FBS with either lo-’ M 1,25(OH)2D3 or ethanol vehicle not exceeding 0.1%. 1,25(OH)2D3 and ethanol-containing media were added to both the top and bottom compartments of the cluster dishes. Media were replaced daily for a total of 4 days. Calcium transport experiments were conducted after 1, 2, 3, and 4 days of 1,25(OH)2D3 and control treatment. 1,25(OH)2D3 concentration of culture media containing 5% FBS was -5 x lo-l2 M. Measurements. Radioactivity was determined by liquid scintillation counting using a Packard ZOOOCA liquid scintillation counter. Calcium transport was expressed
TRANSPORT
IN
CACO-2
CELLS
as nanomoles calcium transported to the serosal compartment per minute per well during the 30- to 60-min time point, unless specified otherwise. Kinetic constants for transport experiments were calculated from a computer-fitted Michaelis-Menten equation modified to include a linear diffusional component. The data at each calcium concentration were weighted by the reciprocal of their variance. The kinetics of calcium transport were determined by fitting the transport rate at various calcium concentrations to the following equation total transport
=
WI + Knax[~l/Kn + [Sl
where C is the nonsaturable linear component, S is the concentration of calcium in the transport buffer, Vmax is the maximal rate of calcium transport of the saturable component, and K, is the buffer calcium concentration that results in half-maximal velocity. Statistics. The statistical significance of treatment effects of calcium transport and calcium kinetic parameters were made using a t test. Data are presented as means t SE. RESULTS
Permeability of Caco-2 cell monolayer. The transport of [3H]inulin, a molecular probe that does not permeate membranes, was assessed in the presence and absence of a cell monolayer to test the permeability of the Caco-2 monolayer grown for 15 days on permeable supports. We found the Caco-2 cell monolayer at day 15 in culture to be essentially impermeable to inulin. Only 0.6% inulin crossed the cell monolayer in a 2-h period. In contrast, 35% diffused across the permeable filters in the absence of a cell layer in the same time period. Kinetics of transepithelial calcium transport. In our initial studies, the rate of transepithelial calcium transport by Caco-2 cells was determined in confluent, differentiated cells grown in media containing 20% FBS until day 15 in culture. We found the transport of calcium into the bottom compartment to be linear up to at least 120 min at calcium concentrations between 0.75 and 15 mM (Fig. 1A). Because transport was linear over this time period, we have estimated the linear transport rate in subsequent experiments using only the 30- and 60-min time points. The concentration dependence of transepithelial calcium transport in Caco-2 cells was determined and found to be nonlinear (Fig. IB), suggesting the presence of a saturable transport component. The data for calcium transport (nmol Ca min-l . well-l) were fitted to the modified Michaelis-Menten equation to estimate the kinetic parameters for the saturable and nonsaturable transport components. The results of three control kinetic experiments conducted in Caco-2 cells that had been grown in culture media supplemented with 20% FBS until day 15 in culture yielded an apparent K, for the saturable transport component of 2.9 t 1 mM, a maximum value for saturable transport ( Vmax)of 1.4 t 0.1 nmol min. well-‘, and a linear nonsaturable transport component (C) of 0.13 t 0.2 nmol~min-l~well-l~mM-l. Effect of 1,25(OH)2D3 on transepithelial calcium transDart. The effect of 1,25(OH)2D3 on calcium transport in l
l
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 9, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
VITAMIN
D-REGULATED
CALCIUM
TRANSPORT
IN
CACO-2
8
hinl
G209
CELLS
4
0
Ca Concentration
(ml41
1. Time course and concentration dependence of calcium transport across monolayers of Caco-2 grown on permeable filter supports for 15 days in culture media containing 20% fetal bovine serum (FBS). A: calcium transport was linear between 5 and 120 min at all calcium concentrations tested between 0.75 and 15 mM. Transport data for 15 mM calcium from a representative experiment are shown for illustration. B: rate of total calcium transport (circle) was estimated from amount of calcium transported to lower compartment between 30 and 60 min after adding 45Ca to top compartment of filter support and plotted against buffer calcium concentration. Data were fitted to modified Michaelis-Menten equation to derive parameters for saturable and nonsaturable calcium transport. n , Saturable calcium transport calculated from this equation; A, nonsaturable transport. Each experimental point represents mean of 3 wells. Data are from 1 representative experiment. Two other experiments were conducted and yielded similar results. In this experiment buffer-calcium concentration that results in half-maximal velocity was 2.9 mM, maximal and nonsaturable linear component rate of calcium transport of saturable components was 1.2 nmol min-’ . well-‘, was 0.16 nmol . min. well-’ mM? FIG.
l
Caco-2 cells was determined in cells grown in media containing 5% FBS from day 11 to 15 in culture in the presence or absence of 10m8 M 1,25(OH)2D3. Calcium transport measured at 7.5 mM calcium in the transport buffer was significantly greater (2.8 t 0.2 vs. 1.2 t 0.1 nmol. min-’ . well-‘, P < 0.001, n = 6 experiments) in cells treated . . with 1,25(OH)zD3 compared with vehicletreated cultures. Effect of 1,25(OH)2D3 concentration on calcium transport. The effect of 1,25(OH)zDs concentration on calcium transport in Caco-2 cells was assessed by incubating cultures for 4 days in media containing 5% FBS in the absence or presence of 10-l’ to 10m7 M 1,25(OH)2D3. The results are illustrated in Fig. 2. With increasing concentrations of 1,25(OH)zD3 there was an apparent dosedependent increase in calcium transport rates. 1,25(OH)2D3 increased calcium transport at 10-l’ M, the lowest concentration tested. In contrast, 10B7 M 25(OH)D3 did not significantly increase calcium transport (data not shown). Time course of 1,25(OH)2D3-induced calcium transport.
Caco-2 cells were cultured until day 11 in media containing 20% FBS. On day 11 the FBS concentration was reduced to 5% and cultures received either IO-’ M 1,25(OH)2D3 or ethanol vehicle for the next 4 days. Calcium transport was measured at 7.5 mM calcium in
the transport buffer after 1,2,3, and 4 days of treatment. Calcium transport was significantly increased by 1,25(OH)zDa after 1 day of treatment (1.9 vs. 2.9 nmol min-’ .well-1, P < 0.005). Longer periods of treatment did not result in further augmentation of the rate of calcium transport in 1,25(0H)zDs-treated cells. An additional study was carried out to investigate whether incubation of cells with 1,25(OH)zD3 for
