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Endocrinology Vol. 141, No. 3 891-900
Copyright © 2000 by The Endocrine Society

ARTICLES

Influence of the in Vivo Calcium Status on Cellular Calcium Homeostasis and the Level of the Calcium-Binding Protein Calreticulin in Rat Hepatocytes1

Geneviève Mailhot, Jean-Luc Petit, Christian Demers and Marielle Gascon-Barré

Centre de Recherche, Hôpital Saint-Luc, Centre Hospitalier de l’Université de Montréal (G.M., J.-L.P., C.D., M.G.-B.), and Départements de Nutrition (G.M., M.G.-B.) and Pharmacologie (M.G.-B.), Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H2X 1P1

Address all correspondence and requests for reprints to: Dr. Marielle Gascon-Barré, Centre de Recherche, Hôpital Saint-Luc, Centre Hospitalier de l’Université de Montréal, 264 René-Lévesque boulevard East, Montréal, Québec, Canada H2X 1P1. E-mail: marielle.gascon.barre{at}umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Little attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate -sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TO DATE LITTLE attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite the fact that chronic states of hypo- or hypercalcemia are often observed in humans at all ages. Pathologies such as rickets, osteoporosis, growth retardation, suboptimal insulin secretion, and nephrolithiasis are only a few of the abnormalities that are induced by perturbations in the in vivo calcium balance. They are associated with a wide range of conditions, such as primary or secondary hyperparathyroidism, malabsorption syndrome, vitamin D (D) deficiency, cancer-induced hypercalcemia, as well as the many disease states induced by mutations in the calcium-regulating hormone receptors and the calcium-sensing receptor that result in inappropriate cellular signaling leading to cellular defects in many tissues and organs (1, 2, 3).

We recently reported that chronic calcium deficiency in vivo perturbs the qualitative and quantitative cytoplasmic Ca2+ concentration ([Ca2+]c) response to several classes of calcium-mobilizing agonists (4, 5, 6, 7). It has also been reported that calcium and/or calcium-regulating hormone deficiency also lead to decreased bile flow (8), inappropriate cellular responses associated with the hepatic regeneration process (6, 9, 10, 11), and the induction of liver foci in a model of hepatocarcinogenesis (12). These observations have contributed to the hypothesis that not only is calcium deficiency in vivo associated with abnormal calcium signaling, but that the cellular calcium pools might also be significantly influenced by chronic hypocalcemia. To date, however, little experimental evidence has been put forward indicating that the hormone-sensitive intracellular Ca2+ pools are significantly sensitive to the in vivo calcium status.

The aim of the present studies was to probe the influence of chronic hypocalcemia on both the resting basal [Ca2+]c and the inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools and to investigate the effect of calcium repletion in the presence or absence of the D3 hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on these pools to further illustrate the participation of the in vivo calcium status in their regulation.

We now report that calcium deficiency secondary to D depletion in vivo leads to significant decreases in the resting [Ca2+]c, the size of the IP3-sensitive Ca2+ pools, and the abundance of one of the major endoplasmic reticulum (ER) calcium-binding protein, perturbations that are all reversible by oral calcium feeding alone, or by repletion with the calcium-regulating hormone 1,25-(OH)2D3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Ionomycin, oligomycin, saponin, rotenone, IP3, thapsigargin (Tg), HEPES buffer, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), EGTA, and sodium pyrophosphate were obtained from Sigma (St. Louis, MO); William’s medium E was obtained from Life Technologies, Inc. (Burlington, Canada); heparin was purchased from Leo Laboratories (Ajax, Canada); BSA, pepstatin, leupeptin, ATP, creatine phosphate, creatine kinase, nitro blue tetrazolium (NBT), and X-phosphate reagents were purchased from Roche Diagnostics (Laval, Canada); fura-2/AM, fura-2-free acid, and mag-fura-2/AM were obtained from Molecular Probes, Inc. (Eugene, OR); nylon and enhanced chemiluminescence membranes, First-Strand complementary DNA (cDNA) synthesis kit was obtained from Amersham Pharmacia Biotech (Baie d’Urfe, Canada); Taq PCR Mix was purchased from QIAGEN (Mississauga, Canada); pCR2.1 was obtained from Invitrogen (Carlsbad, CA); rabbit polyclonal antihuman calreticulin antibody was purchased from Stressgen Biotechnologies Corp. (Victoria, Canada); antirabbit IgG linked to alkaline phosphatase was obtained from PharMingen (San Diego, CA); NBT and X-phosphate reagents were obtained from Roche; polyvinylidene difluoride membrane was obtained from Millipore Corp. (Missisauga, Canada). 1,25-(OH)2D3 was a gift from the Hoffmann-La Roche (Nutley, NJ). All other materials were of analytical grade or better.

Experimental design
The influence of the in vivo calcium status on intracellular Ca2+ homeostasis was studied using hepatocytes isolated (13) from normal male Sprague Dawley rat (Charles River Laboratories, Inc., Canada, Ltd., St. Constant, Canada) livers (N) equilibrated in vitro in culture medium adjusted at a Ca2+ concentration of 1.25 mM, similar to that observed in vivo (serum Ca2+, 1.26 ± 0.02 mM) and in hepatocytes obtained from chronically hypocalcemic rats [Ca-; induced by a functional calcium depletion through D deficiency as previously described (11, 14)] kept in vitro at 0.8 mM extracellular Ca2+ ([Ca2+]e), a concentration similar to the prevailing Ca2+ level in vivo (se Ca2+, 0.80 ± 0.01 mM; P < 0.0001 vs. N). To investigate the reversibility of the calcium deficiency, Ca- rats were replete with either oral calcium alone for 2 weeks with a 3% calcium gluconate solution as drinking water (Ca+) or 1,25-(OH)2D3 at a dose of 28 pmol/day delivered by osmotic minipump for 1 week [1,25-(OH)2D3+] as previously described (11, 14). Hepatocytes obtained from the two latter groups were equilibrated in vitro in culture medium adjusted at the normal [Ca2+]e of 1.25 mM, a concentration comparable to the in vivo Ca2+ (se Ca2+, 1.27 ± 0.01 for Ca+ and 1.28 ± 01 mM for 1,25-(OH)2D3+, respectively).

Hepatocytes were chosen as model cells because their cellular calcium metabolism is well characterized, and they can easily be obtained after in vivo conditioning and used shortly after their isolation procedure under primary culture conditions reflecting closely the in vivo situation, a condition that may be difficult to achieve with other cell types such as osteoblasts or cells in mineralized tissues. In the present studies, [Ca2+]e was, therefore, adjusted to a concentration similar to that observed in vivo so as not to disturb cellular Ca2+ homeostasis, although a previous study clearly indicated that the resting cytoplasmic Ca2+ concentration is not influenced by in vitro [Ca2+]e of 1.25 or 0.8 mM in hepatocytes isolated from hypo- or normocalcemic rats (4).

Concentrations of ionized calcium in whole blood and in culture and experimental solutions were measured with an ICA2 ionized calcium analyzer (Radiometer, Copenhagen, Denmark). All experimental protocols were approved by the institutional animal ethics committee.

Cytoplasmic Ca2+ measurements at the single cell level
Hepatocytes were plated onto collagen-coated coverslips in William’s medium E containing 25 mM bicarbonate, 1% BSA, and 0.8 or 1.25 mM Ca2+, pH 7.4, as mentioned above, at 37 C in 5% CO2 atmosphere. After 60 min of culture, cells were loaded for 30 min at 20 C with 2 µM fura-2/AM in bicarbonate-free William’s medium E supplemented with 2.5% FBS and 1% BSA. Cells were then transferred into a 100-µl plastic chamber to the stage of an inverted microscope (Diaphot, Nikon, Tokyo, Japan) equipped for epifluorescence measurement and superfused (3 ml/min) with a Krebs-Henseleit buffer, pH 7.4, equilibrated with O2-CO2 (95:5, vol/vol) at 32 C. The basal resting cytoplasmic Ca2+ concentration was evaluated at 0.8 (Ca-) or 1.25 mM [Ca2+]e (normocalcemic groups). Tg (5 µM) exposure was achieved in a calcium-free environment containing 0.2 mM EGTA, whereas exposure to phenylephrine (Phe; 5 µM) or La3+ was achieved in a calcium-free environment without EGTA. Test compounds were perfused at a rate of 3 ml/min in Krebs-Henseleit buffer, pH 7.4, equilibrated with O2-CO2 (95:5, vol/vol) at 32 C. Experiments requiring La3+ were carried out under similar conditions, except that a HEPES buffer was used due to the poor solubility of La3+ in Krebs-Henseleit buffer.

Fluorescence signals were obtained from single hepatocytes with an MCID dual excitation spectrofluometer system (Imaging Research, Inc., St. Catherine, Canada) and a refrigerated camera (Hamamatsu Photonics C4880, Hamamatsu, Japan) as imaging device. Excitation wavelengths were 340 and 380 nm, and fluorescence emission was measured at 505 nm. Intracellular dye calibration was performed in situ by perfusion of 2.5 µM ionomycin in a solution containing 4 mM EGTA (Rmin) or 6 mM CaCl2, 10 µM FCCP, and 2.5 µM ionomycin (Rmax). Signal ratios (F350/F380) were transformed into [Ca2+]c according to the method of Grynkiewicz et al. (15). Intracellular dye spectra and loading capacities were equivalent in all groups.

IP3-induced [Ca2+]i mobilization in permeabilized hepatocyte suspension
Two sets of experiments were carried out.

Ca2+ mobilization under steady state conditions. Hepatocytes were incubated at 37 C in the stirred thermostated (37 C) cuvette of a SPEX model CMIT-11I dual excitation spectrofluorometer (Rayonics Scientific, Inc., St. Laurent, Canada). Plasma membranes were permeabilized in medium containing 120 mM KCl, 30 mM HEPES, 1.0 mM MgCl2, 100 µg/ml saponin, 1.0 mM ATP, and an ATP-generating system containing 25 mM creatine phosphate, 25 U/ml creatine kinase, 5 mM sodium pyrophosphate, and 10 µM of the mitochondrial inhibitor FCCP, pH 7.3. Permeabilized cells were washed once, centrifuged, and resuspended in buffer without saponin at a density of 107 hepatocytes/ml according to the method of Missiaen et al. (16). IP3-induced Ca2+ mobilization (1–25 µM) was carried in the presence of 10 µM fura-2 free acid in a calcium-free medium.

Ca2+ mobilization after prior emptying of the IP3-sensitive Ca2+pools. To further evaluate the influence of the in vivo calcium status on the size of the IP3-sensitive Ca2+ pools, hepatocytes were isolated and resuspended in calcium-free medium. They were then stimulated with 5 µM Phe for a period of 30 sec to mobilize IP3-sensitive Ca2+ pools. Cells were then centrifuged, resuspended as described above, and stimulated a second time with 5 µM Phe. After centrifugation, hepatocytes were resuspended at a density of 107 cells/ml. They were then permeabilized, and mobilization of Ca2+ was evaluated using IP3 at doses of 0–25 µM as described above. Permeabilization of cells allowed the IP3-sensitive Ca2+ pools to recapture Ca2+ (buffer Ca2+, 300 nM). To verify the specificity of Ca2+ uptake into cellular pools, replenishment of the IP3-sensitive Ca2+ pools was blocked by application of 10 µM Tg during Phe application as well as during exposure to 300 nM Ca2+.

Influence of in vitro [Ca2+]e on the IP3-sensitive Ca2+pools
To verify whether the cellular Ca2+ pools might be influenced by the in vitro [Ca2+]e, paired hepatocytes from N and from Ca- rats were preincubated at 1.25 or 0.8 mM Ca2+. The cellular Ca2+ pool content was monitor using 5 µM mag-fura-2/AM as a probe according to the procedure of Hofer et al. (17). After mag-fura-2/AM loading (40 min at 20 C), hepatocytes were exposed to 100 µg/ml saponin for a period of 1 min in a calcium-free buffer containing 125 mM KCl, 25 mM NaCl, 10 mM HEPES, 0.1 mM MgCl2, and 1 mM ATP, pH 7.3, at 37 C to achieve plasma membrane permeabilization. Ca2+ mobilization from internal pools was directly achieved with 10 µM IP3 in the buffer described above, but in the absence of saponin. Fluorescence signals were obtained at the single cell level using the imaging system described to measure cytoplasmic Ca2+ concentrations. Data are presented as the signal ratios obtained at 340 and 380 nm.

Store-operated calcium entry
Store-operated/capacitative Ca2+ entry was evaluated in N and Ca- rats by exposing cells to 0.4–3.0 mM extracellular Ca2+ after emptying of the IP3-sensitive calcium pools with two consecutive applications of 5 µM Phe in a calcium-free environment. Ca2+ entry into cells was evaluated in single hepatocytes by monitoring the rise in [Ca2+]c during extracellular Ca2+ application.

Evaluation of the calreticulin gene transcript and protein levels
Total liver RNA was blotted onto nylon membrane and processed for Northern analysis as described previously (18). The radiolabeled probes were: calreticulin, a 1.7-kb rat cDNA fragment obtained by RT-PCR using primers based on the sequence of Murthy et al. (19); and ribosomal 18S RNA, a 1.5-kb human cDNA insert from the EcoRI site of the pBluescript SK-vector (77242, American Type Culture Collection, Manassas, VA). The Primers Software of Williamstone Enterprises (http://www.williamstone.com) was used to generate the calreticulin fragment. Briefly, 5 µg total RNA were converted into cDNA using the First Strand cDNA Synthesis Kit, and 1.0 µl RT reaction was amplified with Taq PCR Master Mix using a Touchdown Thermal Cycler (Hybaid, Teddington, UK). The PCR fragment was inserted into pCR 2.1, and the sequence was confirmed by restriction analysis. The probes were labeled by random oligo-priming (20) using [{alpha}-32P]deoxy-CTP (3000 Ci/mol) and Klenow fragment. Blot hybridization, washing, exposure, and photodensitomeric evaluation were performed as described previously (18).

Relative levels of immunoreactive calreticulin protein were determined by Western blot analysis. Liver samples were homogenized as described by Loyer et al. (21), and protein concentration was determined according to the method of Bradford (22). Twenty micrograms of protein were loaded onto a SDS-PAGE 5–15% acrylamide gradient gel and transferred to polyvinylidene difluoride membranes. The membranes were incubated for 2 h at 37 C with a rabbit polyclonal antihuman calreticulin antibody (1:1000) followed by incubation with an antirabbit IgG linked to alkaline phosphatase (1:1000). The antigen-antibody complex was visualized with nitro blue tetrazolium and X-phosphate reagents. Band quantification was achieved by densitometry scanning (18).

Mathematical and statistical analyses
Results are expressed as the mean ± SEM. Basal resting [Ca2+]c values are, however, presented as cumulative frequency curves and medians due to the right-skewed distribution of the observed [Ca2+]c values. Fitting of data on the observed resting basal cytoplasmic Ca2+ concentration and the IP3-sensitive Ca2+ mobilization from cellular pools was performed according to the nonlinear regression model of Motulsky and Ransnas (23). Statistically significant differences between group means were evaluated by ANOVA or the {chi}2 test as indicated in the figure legends (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Basal cytoplasmic calcium
Figure 1Go presents the basal resting cytoplasmic Ca2+ concentrations in hepatocytes obtained from normal rats as well as from those submitted to a regimen leading to hypocalcemia or after repletion with oral calcium alone or 1,25-(OH)2D3. As illustrated, a highly significant shift to the left in the observed cumulative cytoplasmic Ca2+ concentration frequency curve was observed in cells obtained form hypocalcemic rats. Indeed, the median (cumulative frequency at 50%) resting cytoplasmic Ca2+ value was 115.5 nM compared with a value of 150.5 nM in hepatocytes obtained from normal rats (P < 0.0001). Repletion with 1,25-(OH)2D3 completely normalized the resting cytoplasmic Ca2+ concentration (median, 155 nM), whereas repletion with calcium alone led to a significant increase in basal cytoplasmic Ca2+ (median, 195 nM) not only above that observed in hypocalcemia (P < 0.0001 compared with Ca-) but also above that observed in N or 1,25-(OH)2D3-replete rats (P < 0.0001 compared with either N or 1,25-(OH)2D3+) despite similar circulating Ca2+ concentrations (Ca+, 1.27 ± 0.01; N, 1.26 ± 0.02; 1,25-(OH)2D3+, 1.28 ± 0.01 mM; P = NS).



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  		Figure 1. Influence of the in vivo calcium status on basal resting [Ca2+]c in rat hepatocytes. Hepatocytes were isolated from livers of N, Ca-, Ca+, or 1,25-(OH)2D3+ rats and kept in short term primary culture containing [Ca2+]e similar to that prevailing in vivo. Data show the distribution as cumulative percent frequency of observed [Ca2+]c in basal unstimulated conditions for all preparations studied. N, n = 944 cells obtained from 12 animals; Ca-, n = 817 cells obtained from 12 animals; Ca+, n = 867 cells obtained from 12 animals; 1,25-(OH)2D3+, n = 806 cells obtained from 11 animals. Significant differences among the four sigmoidal cumulative frequency curves was analyzed using nonlinear regression modeling (between-group difference, P < 0.0001). The median (CF50) were 150.5, 115.5, 155, and 195 nM in N, Ca-, 1,25-(OH)2D3+, and Ca+, respectively (N vs. Ca-, P < 0.0001; N vs. 1,25-(OH)2D3+, P = NS; Ca+ vs. Ca-, N, or 1,25-(OH)2D3+, P < 0.0001). Statistically significant differences between group means were evaluated by ANOVA, and individual contrast was determined by the Tukey test. Mean resting [Ca2+]c values were 169.8 ± 3.2, 152.1 ± 3.6, 200.5 ± 4.4, and 221.4 ± 5.0 nM in N, Ca-, 1,25-(OH)2D3+, and Ca+, respectively [main effect, P < 0.001; Ca- vs. N, 1,25-(OH)2D3+ or Ca+, P < 0.01].

 
Evaluation of the hormone-sensitive calcium pools
Response to thapsigargin. In an attempt to probe the cellular Ca2+ pools, three series of experiments were carried out. Hepatocytes were first exposed, in a calcium-free environment, to the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor Tg (25), and the cytoplasmic Ca2+ response was evaluated. Tg exposure prompted increases in [Ca2+]c in all groups, but the Ca2+ peak amplitude was significantly lower in cells obtained from Ca- animals than that observed in hepatocytes obtained from normocalcemic rats (P < 0.0001; Fig. 2Go). Repletion with calcium alone increased cytoplasmic Ca2+ during Tg exposure to a value similar to that observed in N, whereas 1,25-(OH)2D3 repletion led to an increase in [Ca2+]c to a level higher than that observed in hepatocytes obtained from rats supplemented with oral calcium alone. One striking difference among the various groups, however, was the frequency with which Tg induced an elevation in cytoplasmic Ca2+. Indeed, detectable increases in cytoplasmic Ca2+ in response to Tg application were observed in 69% and 62% of the cells obtained from N and Ca+ rats, respectively (P = NS) and in the percentage of cells obtained from 1,25-(OH)2D3-replete rats (P < 0.001 vs. N; P < 0.01 vs. Ca+). By contrast, only 14% of the Ca- animals exhibited a detectable cytoplasmic Ca2+ response in the presence of Tg (P < 0.0001 vs. all other groups). The absence of detectable responses to Tg did not, however, seem to be due to methodological problems, as nonresponding cells were found to be fully responsive to stimulation with 5 µM phenylephrine, or 10 nM vasopressin (data not shown).



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  		Figure 2. Influence of the in vivo calcium status on the amplitude of the initial [Ca2+]c during exposure to Tg in calcium-free buffer. Hepatocytes were isolated from rat livers of N (n = 250 cells obtained from 6 rats), Ca- (n = 242 cells obtained from 8 rats), Ca+ (n = 221 cells obtained from 10 rats), or 1,25-(OH)2D3+ (n = 229 cells obtained from 8 rats) and kept in short-term primary culture containing [Ca2+]e similar to that prevailing in vivo before Tg application. The increases in [Ca2+]c were evaluated in single hepatocytes using the Ca2+-sensitive Ca2+ probe fura-2/AM. The insert represents a typical [Ca2+]c response to Tg exposure in a single hepatocyte obtained from a normal rat hepatocyte. Significant differences between group means were analyzed by ANOVA, with individual between-group comparisons performed using the Tukey test (main effect, P < 0.0001; effect of group, P < 0.0001). Analysis of the number of cells exhibiting a detectable increase in [Ca2+]c (see text) was performed using the {chi}2 test.

 
IP3-mediated Ca2+mobilization. To investigate further the impact of calcium deficiency on the hormone-sensitive calcium pools and to address the short-comings in the Tg experiments, permeabilized hepatocytes were directly probed with increasing concentrations of IP3 under either steady-state conditions (Fig. 3Go) or after Ca2+ mobilization with two successive doses of Phe before IP3 exposure (Fig. 4Go). As shown in Fig. 3AGo, calcium deficiency led to a highly significant decrease in IP3-dependent Ca2+ mobilization compared with that observed in cells obtained from normal rats (P < 0.0001). Repletion with oral calcium or 1,25-(OH)2D3 similarly increased IP3-dependent Ca2+ mobilization to a level slightly above (but not significantly) that observed in cells obtained from normal subjects, but to a level highly above that observed in Ca- (P < 0.0001). Ca2+ mobilization in response to IP3 exposure was completely blocked by heparin, an inhibitor of the IP3 receptor (26, 27) (Fig. 3Go, B and C).



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  		Figure 3. Influence of in vivo calcium status on the IP3-induced Ca2+ mobilization in permeabilized hepatocytes. A, Data present a dose-response curve (1–25 µM) to IP3 in hepatocytes (107/ml) obtained from N, Ca-, Ca+, or 1,25-(OH)2D3+ rats. The Ca2+-sensitive probe fura-2 acid was used to evaluate the Ca2+ mobilized by exposure to increasing concentrations of IP3. Significant differences between group means were analyzed using nonlinear regression modeling: Ca- vs. all other groups, P < 0.0001; difference between normocalcemic groups, P = NS. B, Representative trace illustrating the Ca2+ mobilized after application of 25 µM IP3 in permeabilized hepatocytes. C, Effect of heparin (50 µg/ml) on the mobilization of Ca2+ after 25 µM IP3 application.

 


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  		Figure 4. Influence of the in vivo calcium status on the IP3-induced Ca2+ mobilization in permeabilized hepatocytes after emptying/recharging of the IP3-sensitive Ca2+ pools by two successive 5-µM Phe applications, followed by exposition to 300 nM Ca2+. Data present a dose-response curve (1–25 µM) to IP3 in hepatocytes (107/ml) obtained from N, Ca-, Ca+, or 1,25-(OH)2D3+ rats. The Ca2+-sensitive probe fura-2 acid was used to evaluate the Ca2+ mobilized by exposure to increasing concentrations of IP3. Significant differences between group means were analyzed using nonlinear regression modeling: Ca- vs. all other groups, P < 0.0001; difference between normocalcemic groups, P = NS. B, Representative trace illustrating the Ca2+ mobilized after application of 25 µM IP3 in permeabilized hepatocytes following emptying/replenishment of the IP3-sensitive Ca2+ pools. C, Effect of Tg (10 µM) on the mobilization of Ca2+ after 25 µM IP3 application. Note the absence of effect of Tg on Ca2+ mobilization induced by the calcium ionophore ionomycin.

 
As illustrated in Fig. 4AGo, prior emptying of the IP3-sensitive Ca2+ stores with two successive Phe applications followed by acute replenishment of the pools in the presence of 300 nM Ca2+ also led to a highly significant decrease in IP3-dependent Ca2+ mobilization in hepatocytes obtained from calcium-deficient animals compared with that observed in cells obtained from all other normocalcemic rats (P < 0.0001). Repletion with oral calcium or 1,25-(OH)2D3 increased IP3-dependent Ca2+ mobilization to a level similar to that observed in cells obtained from normal animals. As illustrated in Fig. 4Go, B and C, prevention of the IP3-sensitive Ca2+ pool replenishment with Tg completely blocked IP3-induced Ca2+ mobilization, whereas Ca2+ mobilization by the calcium ionophore ionomycin remained fully operational.

Influence of the in vitro [Ca2+]e. The influence of the in vitro extracellular Ca2+ concentration on the IP3-sensitive Ca2+ pools is presented in Fig. 5Go. The data clearly show that, under our experimental conditions, preincubation of cells from either normal (Fig. 5AGo) or calcium-deficient (Fig. 5BGo) rat livers at 0.8 or 1.25 mM [Ca2+]e does not influence the cellular Ca2+ pool content in either the resting state (before IP3 application) or after IP3-induced Ca2+ mobilization.



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  		Figure 5. Evaluation of the Ca2+ content of the IP3-sensitive pools under in vitro incubation of 0.8 () or 1.25 mM Ca2+ () in hepatocytes obtained from the same rat liver. The mean signal ratios of the mag-fura-2 Ca2+-sensitive probe was obtained before as well as after IP3 application. A, Normocalcemics (n, hepatocytes obtained from three rats; before IP3 application, n = 119 hepatocytes; after IP3 application, n = 67 hepatocytes). B, Hypocalcemics, hepatocytes obtained from three rats (before IP3 application, n = 110 hepatocytes; after IP3 application, n = 52 hepatocytes). Statistically significant differences between group means were analyzed by Student’s t test.

 
Store-operated calcium entry
To study whether a store-operated/capacitative Ca2+ entry mechanism(s) (17, 28) might be responsible for the relative depletion state of the IP3-sensitive calcium pools in hypocalcemia, Ca2+ entry during exposure to [Ca2+]e after emptying of the IP3-sensitive calcium pools with two consecutive applications of 5 µM phenylephrine in a calcium-free environment was investigated in hepatocytes obtained from either N or Ca- rats. As illustrated in Fig. 6Go, A and B, Ca2+ entry from the external compartment was not perturbed by hypocalcemia and was blocked in both N and Ca- by La3+ (Fig. 6Go, C and D), a known blocker of the store-operated calcium channel (29).



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  		Figure 6. Influence of the in vivo calcium status on store-operated/capacitative Ca2+ entry in hepatocytes obtained from N or Ca- rats. In both groups, hepatocytes were kept in short term culture containing [Ca2+]e similar to that prevailing in vivo before the Ca2+ entry studies. Hepatocytes were stimulated with two successive dose of 5 µM Phe to empty the IP3-sensitive Ca2+ stores and then were exposed to [Ca2+]e. The store-operated/capacitative Ca2+ entry was monitored by evaluating the increase in [Ca2+]c during exposure to [Ca2+]e using the fura-2/AM calcium-sensitive probe in single hepatocytes (A and B). In hepatocytes obtained from both groups, Ca2+ entry during exposure to [Ca2+]e after emptying of the IP3-sensitive Ca2+ pools by 5 µM Phe could be blocked by exposure to 2.0 mM La3+ (C and D).

 
Calreticulin gene transcript and protein levels
As illustrated in Figs. 7Go, A and B, calcium depletion significantly lowered the steady state expression of the calreticulin gene transcript (P < 0.01 vs. N), whereas repletion with calcium alone or 1,25-(OH)2D3 significantly increased the calreticulin gene transcript to a level comparable to that observed in N. The calreticulin protein levels (Fig. 7BGo) were also significantly lower in cells isolated from Ca- than in those obtained from N (P < 0.0001). Repletion with 1,25-(OH)2D3 or calcium alone contributed to significantly increase CRT levels above those observed in Ca- (P < 0.001 vs. 1,25-(OH)2D3+; P < 0.002 vs. Ca+), levels that were not significantly different from those in hepatocytes isolated from normal animals.



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  		Figure 7. Influence of the in vivo calcium status on the steady state expression of the calreticulin gene transcript (A) and the immunoreactive protein level (B) in rat liver. A, Fifteen micrograms of total liver RNA were submitted to Northern analysis as indicated in the text. To normalize for unequal loading of RNA to the gel, the filter was stripped and rehybridized to the ribosomal 18S [{alpha}-32P]cDNA probe. Representative Northern blot analysis from a single animal from each group is shown. The numbers of rats used were: N, n = 10; Ca-, n = 6; Ca+, n = 5; and 1,25-(OH)2D3+, n = 6. B, Twenty micrograms of protein were submitted to Western analysis as described in the text. A representative Western blot from a single animal from each group is shown (N, n = 6; Ca-, n = 5; Ca+, n = 7; 1,25-(OH)2D3+, n = 6). All significant differences between group means in A and B were analyzed by ANOVA, with individual contrasts evaluated by the Fisher test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Due to the large concentration gradient between extra- and intracellular Ca2+, basal resting cytoplasmic Ca2+ is largely considered to be insensitive to the prevailing [Ca2+]e. However, to date, most studies have evaluated the impact of external Ca2+ in in vitro models, most often using Ca2+ concentrations well outside the normal physiological range in cells or cell lines maintained in vitro for extended periods of time. In contrast, the present data were obtained using cells isolated from animals subjected to a well characterized protocol of calcium deficiency secondary to D depletion. In addition, great care has been taken to maintain cells in vitro at [Ca2+]e similar to those prevailing in vivo and to keep the ex vivo period as short as possible so as not to disturb the in vivo conditioned cellular Ca2+ homeostasis, although under short term incubation conditions, [Ca2+]c has been shown not to be disturbed by in vitro Ca2+ variations in the range of 0.8–1.25 mM (4). The data obtained under these conditions clearly show that hypocalcemia significantly lowers the basal resting cytoplasmic Ca2+ concentration. Our observations, therefore, indicate that in otherwise normal animals (11, 30), calcium deprivation is able to shift to the left the resting hepatocellular calcium concentration curve as well as the mean [Ca2+]c compared with the concentration observed in normal animals. Moreover, this shift was shown to be fully reversible by calcium repletion in association with the D hormone or by oral calcium repletion alone, the latter condition leading to an apparently supranormal correction in the resting cytoplasmic Ca2+ concentration. The reason for the observation that the resting hepatocytic [Ca2+]c was consistently higher in cells obtained from animals supplemented with oral calcium alone despite circulating Ca2+ concentrations similar to those in the two other normocalcemic groups is not presently known. However, the level of dietary calcium needed to maintain normal circulating Ca2+ concentration was much higher in the Ca+ group (3% calcium gluconate in drinking water) than in the N (no added calcium in drinking water) and 1,25-(OH)2D3+ groups (0.5% calcium gluconate in drinking water). Hence, dietary calcium per se might be an important contributing factor to the resting [Ca2+]c. Alternatively, it could be argued that 1,25-(OH)2D3 and/or PTH may also be contributing factors, as Ca+ animals remained D-deplete despite normalization of circulating Ca2+, a state that is known to not completely normalize the secondary hyperparathyroidism induced by D depletion (31). The data obtained, hence, illustrate the impact of the in vivo Ca2+ status on the resting cellular Ca2+ homeostasis, but, most importantly, they raise the question as to the impact of the in vivo calcium status, with its observed effect on the resting cytoplasmic Ca2+ concentration, on the state of the intracellular calcium pools.

Both series of studies aimed at probing the size of the hormone-sensitive Ca2+ pools strongly suggest the presence of a significant reduction in the apparent content of these pools. In the Tg experiments, the absence of a Tg-induced elevation in [Ca2+]c in a significant number of cells may have been due to several variables, such as a low Ca2+ pool content, a rapid Ca2+ uptake by adjacent organelles such as the mitochondria (32), or a lower Tg-induced passive Ca2+ leak from the ER in some groups [i.e. Ca- or 1,25-(OH)2D3+] than in others (33). The latter two conditions would lead to a significant underestimation of the size of the Tg-sensitive calcium pools in the affected groups. However, the IP3 mobilization studies circumvent the weaknesses of the Tg studies by directly probing the hormone-sensitive pools with IP3. These studies clearly demonstrate that under both steady state conditions and after emptying/replenishment, the IP3-dependent Ca2+ pools were significantly lowered by the hypocalcemic condition and fully restored by normalizing serum Ca2+ with calcium alone or 1,25-(OH)2D3. The reason for the consistently higher Ca2+ mobilization under conditions of emptying/replenishment of the IP3-sensitive Ca2+ pools than under steady state conditions is not known, but is postulated to be due to either a greater sensitivity of the IP3 receptor induced by the prior exposure to Ca2+, or to an increase in the availability of Ca2+ after its acute uptake into cellular pools. Here again, the in vitro incubation conditions (0.8 and/or 1.25 mM Ca2+) were shown not to influence pool size, as clearly indicated using mag-fura-2 as a probe to evaluate the IP3-sensitive Ca2+ pool content. These data, thus, clearly indicate that in rat hepatocytes the in vivo calcium status is a major determinant of the size of the IP3-mobilizable Ca2+ pool, as the IP3 concentrations used led to receptor saturation and were within a range able to mobilize both the sensitive as well as the less sensitive Ca2+ units (34, 35).

The mechanisms by which the in vivo calcium status is involved in the regulation of cellular calcium homeostasis is not presently known, as circulating Ca2+ has been well documented not to freely enter cells. However, Ca2+ has been shown to enter cells after agonist stimulation or emptying of the IP3-sensitive calcium pools through store-operated or capacitative calcium channels, a mechanism aimed at replenishing calcium pools after agonist stimulation (17, 28). The data obtained during the present studies indicating that Ca2+ entry after Phe exposure was totally operational in cells obtained from hypocalcemic animals indicate that the mechanism(s) responsible for Ca2+ entry after agonist stimulation, the capacitative calcium entry, is not intrinsically perturbed by calcium deficiency. Furthermore, our studies also clearly show that Ca2+ not only enters the cytoplasmic compartment, but also accumulates in cellular stores, as indicated by the IP3-induced Ca2+ mobilization after emptying/recharging of the hormone-sensitive pools, a recharging that was completely blocked by Tg. These observations indicate that in calcium deficiency, cells can adequately take up and accumulate Ca2+ in cellular pools. However, optimum pool replenishment may be limited by the available concentration of extracellular Ca2+, which in hypocalcemia is significantly lower than in normocalcemia. In addition to Ca2+ entry, cellular Ca2+ homeostasis is regulated by its extrusion at the plasma membrane. A previous study from our laboratory has shown that the plasma membrane Ca2+-adenosine triphosphatase, which is responsible for Ca2+ extrusion from the cell, is also unperturbed by calcium and/or D depletion (4). Collectively, these observations indicate that both Ca2+ entry and calcium extrusion are not the mechanisms responsible for the decrease in the size of the hormone-sensitive Ca2+ pools associated with calcium deficiency, although, as mentioned above, the level of [Ca2+]e may, per se, be a limiting factor, particularly during long lasting calcium deficiency.

In light of the data indicating the presence of lower cellular Ca2+ pools in calcium deficiency, it was postulated that cellular calcium accumulation in hormone-sensitive pools might involve a protein(s) known to bind Ca2+ within the ER, which has been reported to be able to modulate the functional size of the cellular calcium pools (36, 37, 38). Lately, several laboratories have published data indicating that calreticulin, a high capacity, low affinity calcium-binding protein (CaBP) located in the lumen of the ER, is able to play such a role (38, 39, 40, 41). To determine whether calreticulin might be influenced by hypocalcemia, investigation of the steady state expression of its gene transcript as well as associated protein was carried out in hepatocytes from the four experimental groups. The data obtained indicate that chronic hypocalcemia is accompanied not only by a lowering of the IP3-dependent Ca2+ pools but also by a significant, but reversible, decrease in the calreticulin gene transcript. Moreover, its immunoreactive protein level was normalized by the repletion protocols at the time point studied during the present experiments. Ongoing studies in our laboratory (unpublished) show that the calreticulin gene transcript rapidly responds to dietary calcium, suggesting a possible regulation by calcium availability.

The data illustrate that calreticulin, a major luminal CaBP known to be mainly located in Tg- and IP3-sensitive calcium stores (39, 40, 42) and suspected to act as a major regulator of the size of the ER hormone-sensitive calcium pools, is significantly influenced by the in vivo calcium status (37, 39, 40). In addition, the latter observation suggests that restoring the in vivo calcium status by oral calcium alone is sufficient to normalize the level of the ER CaBP calreticulin, as judged by the levels of its messenger RNA and immunoreactive protein. The predominant role of calcium in the modulation of the cellular calcium pools in the present model, as judged by the size of the functional IP3-sensitive Ca2+ pools, is congruent with the reported low level of nuclear vitamin D receptor (VDRn) in hepatocytes (43, 44, 45). Interestingly, preliminary data from our laboratory also indicate that similar effects on cellular calcium pools are observed by varying the [Ca2+]e alone in the VDRn-positive osteoblastic cell line ROS 17/2.8 (46), suggesting that the data obtained for hepatocytes during the present studies may well reflect the influence of calcium status on cells known to be targets for the calcium/vitamin D endocrine system.

In conclusion, the five criteria chosen to evaluate the impact of the extracellular calcium status on cellular calcium homeostasis clearly illustrate that a state of calcium deficiency in vivo significantly alters cellular calcium homeostasis by lowering both the basal [Ca2+]c as well as the IP3-dependent Ca2+ pools, a lowering that cannot be attributed to perturbations in the mechanisms responsible for Ca2+ entry through the store-operated calcium channels or extrusion of Ca2+ at the plasma membrane as reported previously (4). The decrease in the size of the cellular calcium pool was, however, shown to be accompanied by a significant decrease in the steady state levels of the calreticulin transcript as well as its protein, which were both shown to promptly respond to oral calcium repletion alone independently of 1,25-(OH)2D3 administration. Collectively, these data, which were obtained using a well characterized physiological model of calcium deficiency in otherwise normal rats, illustrate that the in vivo calcium status is a main determinant of cellular calcium, an idea that gives rise to a paradigm shift in our understanding of cellular calcium homeostasis and regulation. These observations may contribute to our understanding of several pathologies associated with suboptimal calcium status, particularly in the context of the aging population, in which poor calcium and D status is often reported (47, 48, 49, 50), and in the investigation of its role in the development of debilitating diseases, such as osteoporosis, inappropriate insulin secretion, and others (50, 51, 52, 53, 54).


   Acknowledgments
 
The authors are grateful to Ms. Manon Livernois for her excellent secretarial assistance.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada. Back

Received September 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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