Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Almadén, Y.
Right arrow Articles by Rodríguez, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Almadén, Y.
Right arrow Articles by Rodríguez, M.
J Am Soc Nephrol 13:693-698, 2002
© 2002 American Society of Nephrology

Regulation of Arachidonic Acid Production by Intracellular Calcium in Parathyroid Cells: Effect of Extracellular Phosphate

Yolanda Almadén*, Antonio Canalejo{dagger}, Evaristo Ballesteros{ddagger}, Gracia Añón*, Sagrario Cañadillas* and Mariano Rodríguez*

*Research Unit and Nephrology Service, Reina Sofia University Hospital, Cordoba, Spain; {dagger}Department of Environmental Biology and Public Health, University of Huelva, Huelva, Spain; and {ddagger}Department of Physical and Analytical Chemistry, Technical University School of Linares, University of Jaen, Jaen, Spain.

Correspondence to Dr. Mariano Rodriguez, Unidad de Investigacion, Hospital Universitario Reina Sofia, Avda Menendez Pidal s/n, Cordoba 14004, Spain. Phone: 957-217242; Fax: 957-202542; E-mail: mrodriguez{at}sofia.hrs.sas.cica.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The action of extracellular calcium on the calcium receptor in parathyroid cells results in activation of phospholipase C (PLC), PLD, and PLA2. The PLA2-arachidonic acid (AA) intracellular signaling pathway mediates inhibition of parathyroid hormone (PTH) secretion. In addition, stimulation of the calcium receptor produces increases in intracellular calcium levels. It was demonstrated that high extracellular phosphate levels reduce the production of AA, a mechanism by which phosphate may stimulate PTH secretion. The objective was to determine, in parathyroid tissue, whether AA production is stimulated by increases in intracellular calcium levels and to investigate whether the decreased AA production induced by high extracellular phosphate levels could be modified by increases in intracellular calcium levels. Experiments were performed in vitro using parathyroid tissue. The intracellular calcium level was increased by incubation with an ionophore (A23187), which increases calcium influx across the cell membrane, or thapsigargin, which releases calcium from intracellular stores. The phosphate concentration in the medium was normal (1 mM) or high (4 mM). The response to calcium was evaluated by incubation with 0.6 or 1.35 mM calcium concentrations. AA production by parathyroid tissue was measured by gas chromatography. In parathyroid tissue incubated with either a calcium ionophore or thapsigargin, there was an increase in AA production, together with inhibition of PTH secretion, suggesting that PLA2 is activated by the elevation in intracellular calcium levels. Therefore, the effect of intracellular calcium level elevation on AA production in the presence of high extracellular phosphate levels was evaluated. The results demonstrate that, despite high phosphate levels in the medium, both the ionophore and thapsigargin were capable of inducing a marked increase in AA production, which was associated with a decrease in PTH secretion. In conclusion, in parathyroid tissue, AA levels can be regulated by an ionophore and thapsigargin, both of which increase cytosolic calcium concentrations. The stimulation of PTH secretion by high phosphate levels can be prevented by increases in intracellular calcium levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extracellular calcium regulates parathyroid hormone (PTH) secretion by acting on a G protein-coupled calcium-sensing receptor (CaR) (1). The intracellular signaling pathways involved in the regulation of PTH secretion include activation of phospholipase C (PLC), PLD (2), and PLA2 (3–5).

High extracellular calcium levels stimulate parathyroid cell PLA2 activity, resulting in increased production of arachidonic acid (AA), the substrate for the 12- and 15-lipoxygenase pathways, which have been directly implicated in the inhibition of PTH release (4). The precise mechanisms by which high extracellular calcium levels stimulate PLA2 activity in parathyroid cells are not completely clear, although recent work suggests that mitogen-activated protein (MAP) kinases are involved in PLA2 activation (6). In other cells, the activity of PLA2 is dependent on intracellular calcium levels (7). In parathyroid cells, the intracellular calcium level increases in response to calcium receptor-dependent PLC activation; however, it is not known whether elevation of intracellular calcium levels stimulates PLA2 activity, which could be a reasonable mechanism by which PLA2 activity is coupled to the activation of PLC.

Work by us and others (8–15) has demonstrated that, independent of the calcium concentration, high extracellular phosphate levels directly stimulate PTH secretion and synthesis, which emphasizes the importance of phosphate in the pathogenesis of secondary hyperparathyroidism (16). Furthermore, we demonstrated in a recent work that high extracellular phosphate levels reduce the production of AA by parathyroid tissue; this is a mechanism by which phosphate may stimulate PTH secretion (17).

The aim of this study was to determine whether AA production is stimulated by an increase in intracellular calcium levels in parathyroid tissue. If this were to be the case, we were interested in determining whether the low AA production induced by high extracellular phosphate levels could be modified by increases in intracellular calcium levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Parathyroid Tissue
The study was performed with parathyroid glands obtained from male Wistar rats weighing 170 to 200 g. The rats were receiving a 0.6% calcium/0.6% phosphorus diet supplemented with 100 IU/100 g vitamin D. The animals were anesthetized with pentobarbital (50 mg/kg) and, within 2 min after the blood was drained by aortic puncture, the parathyroid glands were dissected free of the thyroid glands, with a dissecting microscope, and removed. Some experiments required a relatively large amount of tissue, in which case dog parathyroid glands were used. These glands were obtained from euthanized, 2- to 5-yr-old, mongrel dogs (13 to 20 kg) that had been donated by the Center for Animal Control. These dogs were healthy and ingested a standard chow diet. The time required to complete the parathyroidectomy in dogs was approximately 3 min.

Incubation Conditions
Intact rat parathyroid glands or small (1-mm3) pieces of dog parathyroid tissue were placed in individual wells, containing 2 ml of incubation medium, resting inside a nylon basket; the glands were maintained at 37°C, with constant rocking and shaking motions (AOS-0; SBS Instruments SA, Badalona, Spain). Cell viability was >80%.

The incubation medium was buffered (pH 7.4) and contained 125 mM NaCl, 5.9 mM KCl, 0.5 mM MgCl2, 1 mM sodium pyruvate, 4 mM glutamine, 12 mM glucose, 25 mM Hepes, 0.1 IU/ml human insulin, 0.1% bovine serum albumin, 100 IU/ml penicillin G, and 100 mg/ml streptomycin. Phosphate concentrations of 1 and 4 mM were obtained by adding NaH2PO4 and Na2HPO4 in 1:2 proportion. CaCl2 was added to yield 0.6, 1.25, or 1.35 mM (as measured with a selective electrode, model 634; Ciba Corning, Essex, England) (8). Measurements of ionized calcium concentrations before and after a 1-h incubation with 1 or 4 mM phosphate demonstrated no change in the ionized calcium concentration. Similarly, magnesium concentrations in the medium, as measured with a colorimetric assay (magnesium kit; Sigma Chemical Co., St. Louis, MO), were unchanged after a 1-h incubation with 1 or 4 mM phosphate. All chemical products were obtained from Sigma.

Effects of High Intracellular Calcium Levels (Induced by Ionophore or Thapsigargin) on AA Production and PTH Secretion
This set of experiments was performed with dog parathyroid tissue, because the quantification of AA by gas chromatography requires a relatively large amount of tissue. In previous in vitro work, we demonstrated that the PTH response to calcium and phosphate in dog parathyroid tissue was similar to that observed in intact rat parathyroid glands (17). Dog parathyroid tissue was incubated for 30 min each with low (0.6 mM) and high (1.35 mM) calcium concentrations, with or without ionophore A23187 (10 µM) or thapsigargin (1 µM) (both from Sigma) added to the medium. Both compounds produce increases in cytosolic calcium levels; ionophores increase the calcium influx across the cell membrane, whereas thapsigargin acts through the release of calcium from intracellular stores (18). In preliminary experiments, we determined that 10 µM was the minimal concentration of ionophore needed to inhibit the PTH secretion stimulated by low calcium levels (0.6 mM) (1, 5, and 10 µM A23187 produced decreases in PTH secretion of 20, 60, and 98%, compared with the control values obtained with 0.6 mM calcium with vehicle added). Thapsigargin at 1 µM has been demonstrated to prevent endoplasmic reticulum calcium transport (19). After completion of the incubation period, parathyroid tissue was homogenized, with a glass-glass homogenizer, in a solution containing 50 mM Tris-HCl (pH 7.5 at 25°C), 0.2 mM ethylenediaminetetraacetate, and 0.5 mM dithiothreitol. AA was quantified by gas chromatography, as described elsewhere (20) (model 5890-A; Hewlett Packard, Avondale, PA); with this method, the coefficient of variation for AA quantification was 2.5%. In parallel experiments, dog parathyroid tissue was incubated for 1 h and the PTH concentration in the medium was measured by using a human intact PTH Immunoradiometric Assay (IRMA) kit (Nichols Institute, San Juan Capistrano, CA) (17). AA and PTH values were expressed relative to the protein content of the sample, which was measured by using the method described by Bradford (21).

Effects of High Intracellular Calcium Levels (Induced by Ionophore or Thapsigargin) on AA Production with a High Extracellular Phosphate Concentration
These experiments were designed to determine whether high intracellular calcium levels correct the reduced production of AA induced by high extracellular phosphate levels. The experiments were performed with dog parathyroid tissue, using the protocol described above. The effects of ionophore (10 µM) and thapsigargin (1 µM) were tested with low and high calcium concentrations, with both normal (1 mM) and high (4 mM) phosphate concentrations in the medium.

Effects of High Intracellular Calcium Levels (Induced by Ionophore or Thapsigargin) on PTH Secretion with a High Extracellular Phosphate Concentration
Intact rat parathyroid glands were incubated for 1 h in 1.25 mM calcium, for determination of baseline production of PTH; the glands were then transferred, for an additional 2 h, to a medium containing either a low (0.6 mM) or high (1.35 mM) calcium concentration. The ionophore A23187 (10 µM) or thapsigargin (1 µM) was added to the medium. The experiments were performed using normal (1 mM) or high (4 mM) phosphate concentrations in the medium. The incubation medium was replaced every hour, and the PTH concentration in the medium was measured by using a rat intact PTH IRMA kit (Nichols Institute). The intra- and interassay coefficients of variation were 4.3 and 4.7%, respectively. The protein content of the tissue sample was determined by using the method described by Bradford (21).

Statistical Analyses
Differences between more than two means were evaluated by ANOVA followed by the Duncan test. Paired or unpaired t tests were used to compare two means from the same group or different groups of glands, respectively. The results are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of High Intracellular Calcium Levels on AA Production and PTH Secretion
AA production by dog parathyroid tissue was greater in high calcium (1.35 mM) than low calcium (0.6 mM) concentrations (P < 0.01) (Figure 1A) and, as expected, PTH secretion was decreased in high calcium conditions, compared with low calcium conditions (P < 0.01) (Figure 1B). In parathyroid tissue incubated with a low calcium concentration, the addition of calcium ionophore (A23187, 10 µM) resulted in an increase in AA production to a value that was similar to that for the high calcium concentration. This increase in AA production was associated with a decrease in PTH secretion to a level similar to that observed with a high calcium concentration (Figure 1). The addition of thapsigargin (1 µM) to the medium with a low calcium concentration produced changes in AA and PTH similar to those observed with the ionophore.



View larger version (26K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Effects of ionophore (I) A3187 (10 µM) and thapsigargin (T) (1 µM) on arachidonic acid (AA) production (A) and parathyroid hormone (PTH) secretion (B) by dog parathyroid tissue. The phosphate concentration in the medium was 1 mM for all groups. Ca, calcium. Values are means ± SEM (n = 6 for the 0.6 mM plus thapsigargin group, n = 9 for the other groups). aP < 0.01 versus all other groups in the same panel.

 
Effects of High Cytosolic Calcium Levels on the Regulation of AA Production by Phosphate
In medium containing 1.35 mM calcium, the increase in the phosphate concentration from 1 to 4 mM reduced by one-half the production of AA by dog parathyroid tissue (Figure 2A). The addition of either ionophore or thapsigargin to the high-calcium/normal-phosphate medium produced no effect on AA production, suggesting that AA synthesis is maximally stimulated with high calcium levels. With the same high calcium concentration and a phosphate concentration of 4 mM, both the ionophore and thapsigargin induced significant increases in the production of AA, to values that were not significantly different from the control values obtained with 1 mM phosphate.



View larger version (46K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. AA production by dog parathyroid tissue incubated with high (A) or low (B) calcium (Ca) concentrations. Experiments were performed with 1 or 4 mM phosphate (P) concentrations, with or without addition of 10 µM ionophore (A23187) or 1 µM thapsigargin. Values are means ± SEM (n = 6 for the two thapsigargin groups, n = 10 for the other groups). aP < 0.05 versus all other columns in A. bP < 0.05 versus ionophore and thapsigargin groups in B.

 
In medium containing 0.6 mM calcium, the production of AA was similarly reduced with 1 and 4 mM phosphate concentrations (Figure 2B). Despite the low calcium concentration in the medium, the addition of ionophore increased AA production not only with 1 mM phosphate but also with 4 mM phosphate. Similarly, the addition of thapsigargin increased AA levels with 1 and 4 mM phosphate. The increases in AA production induced by both ionophore and thapsigargin were similar with 1 and 4 mM phosphate concentrations and reached the levels observed with the high-calcium medium (Figure 2B).

Effects of High Cytosolic Calcium Levels on the Regulation of PTH Secretion by Phosphate
General Protocol.
Because PTH secretion is mainly regulated by extracellular calcium, the experiments were performed with both high (1.35 mM) and low (0.6 mM) calcium concentrations. A calcium concentration of 1.25 mM, which is the normal calcium concentration in rats, was used to evaluate the basal PTH secretion rate. Therefore, after a 1-h incubation with 1.25 mM calcium, the same rat parathyroid glands were incubated for two additional 1-h periods with either 1.35 or 0.6 mM calcium.

Experiments with a High Calcium Concentration in the Medium.
In a medium with a normal phosphate concentration (1 mM), the increase in the serum calcium concentration from 1.25 to 1.35 mM produced no decrease in PTH secretion (Figure 3A). The rate of PTH secretion remained stable during the 2-h experiment. The addition of ionophore or thapsigargin to the 1.35 mM calcium medium produced no further decrease in PTH secretion. This suggests that, with a calcium concentration of 1.35 mM, the corresponding increase in intracellular calcium levels results in maximal inhibition of PTH secretion and further elevation of the intracellular calcium concentration with the addition of ionophore or thapsigargin does not result in a further reduction of PTH secretion.



View larger version (22K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Effects of ionophore (A23187) (10 µM) or thapsigargin (1 µM), compared with control conditions, on PTH secretion by rat parathyroid glands incubated with a high calcium (Ca) concentration (1.35 mM), with normal (1 mM) (A) or high (4 mM) (B) phosphate (P) concentrations. Intact rat parathyroid glands were incubated for 1 h with 1.25 mM calcium (basal), and the same glands were then incubated for an additional 2 h with 1.35 mM calcium. Values are means ± SEM (n = 6 for the two thapsigargin groups, n = 11 for all other groups). aP < 0.05 versus the same calcium concentration without ionophore or thapsigargin added. bP < 0.05 versus a phosphate concentration of 1 mM (shown in A).

 
The results of experiments performed with 4 mM phosphate are presented in Figure 3B. PTH secretion with 4 mM phosphate and 1.25 mM calcium was greater than that with 1 mM phosphate and the same calcium concentration (179 ± 11 versus 97 ± 12 pg/µg protein per h, P < 0.002) (Figure 3). This finding confirms previous results reported by us and others, which demonstrated a direct effect of phosphate on PTH secretion. Increasing the calcium concentration from 1.25 to 1.35 mM produced no significant reduction in PTH secretion. In contrast, the addition of either ionophore or thapsigargin to the medium with 1.35 mM calcium produced a marked decrease in PTH secretion, despite the high phosphate concentration. The PTH values observed with high phosphate levels after a 2-h incubation with ionophore or thapsigargin were similar to those observed with normal phosphate concentrations (Figure 3).

Experiments with a Low Calcium Concentration in the Medium.
A decrease in the calcium concentration from 1.25 to 0.6 mM in a medium with a normal phosphate concentration (1 mM) produced a significant increase in PTH secretion. Both the ionophore and thapsigargin prevented the stimulation of PTH secretion by the low calcium concentration (Figure 4A).



View larger version (21K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Effects of ionophore (A23187) (10 µM) or thapsigargin (1 µM), compared with control conditions, on PTH secretion by rat parathyroid glands incubated with a low calcium (Ca) concentration (0.6 mM), with normal (1 mM) (A) or high (4 mM) (B) phosphate (P) concentrations. Intact rat parathyroid glands were incubated for 1 h with 1.25 mM calcium (basal), and the same glands were then incubated for an additional 2 h with 0.6 mM calcium. Values are means ± SEM (n = 6 for the thapsigargin groups, n = 11 for all other groups). aP < 0.05 versus the same calcium concentration without ionophore or thapsigargin added. bP < 0.05 versus a phosphate concentration of 1 mM (shown in A).

 
The results of experiments performed with 4 mM phosphate are presented in Figure 4B. As previously demonstrated in Figure 3, the change in the phosphate concentration from 1 to 4 mM, with maintenance of the same calcium concentration of 1.25 mM, resulted in an increase in PTH secretion (Figure 4). Reduction of the calcium concentration from 1.25 to 0.6 mM produced an additional, although moderate, increase in PTH secretion (from 213 ± 17 to 239 ± 15 pg/µg protein per h, P < 0.05 by paired t test) (Figure 4B). The PTH values measured with 4 mM phosphate and 0.6 mM calcium were significantly greater than those observed with a normal phosphate concentration (1 mM) and the same low calcium concentration (Figure 4). Similar to results observed with 1 mM phosphate, the addition of either ionophore or thapsigargin to medium containing 4 mM phosphate not only prevented the stimulation of PTH secretion by low calcium levels but also reduced PTH secretion below the values obtained with 1.25 mM calcium (Figure 4B). Therefore, despite the high phosphate concentration, 2-h incubations with ionophore or thapsigargin decreased PTH secretion to 119 ± 18 and 125 ± 23 pg/µg protein per h, respectively (Figure 4B); these values are similar to the PTH secretion observed with a calcium concentration of 1.25 mM and a normal phosphate concentration (1 mM), without ionophore (101 ± 10 pg/µg protein per h) (Figure 4A).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was designed to determine whether AA production by parathyroid cells is stimulated by an increase in intracellular calcium levels and to evaluate the effects of an increase in intracellular calcium levels on AA production and PTH secretion when cells are stimulated by high extracellular phosphate levels. To increase cytosolic calcium concentrations, the parathyroid tissue was incubated with an ionophore that increases calcium influx across the cell membrane and, in separate experiments, with thapsigargin, which increases the release of calcium from intracellular stores. The results of this study demonstrate that, in rat parathyroid tissue incubated with either the calcium ionophore or thapsigargin, there is an increase in AA production, together with inhibition of PTH secretion; therefore, the data suggest that PLA2 is activated by an elevation of intracellular calcium levels. Because in previous studies we demonstrated that AA production by parathyroid cells was decreased by high extracellular phosphate levels (17), we evaluated the effect of an elevation of the intracellular calcium concentration on AA production in the presence of high extracellular phosphate levels. The results of this study demonstrate that, despite high phosphate levels in the medium, both the ionophore and thapsigargin were capable of inducing a marked increase in AA production, which was associated with a decrease in PTH secretion. These findings suggest that high extracellular phosphate levels prevent an appropriate elevation in intracellular calcium levels in response to an increase in extracellular calcium levels.

This study was performed in vitro, using intact parathyroid tissue incubated in media with low (0.6 mM) or high (1.35 mM) calcium concentrations and normal (1 mM) or high (4 mM) phosphate concentrations. We demonstrated in a previous study, using the same methods, that 0.6 mM calcium produces maximal stimulation of PTH secretion and 1.35 mM calcium maximally inhibits PTH secretion (8). Higher concentrations of calcium do not further reduce PTH secretion and may precipitate calcium when used with 4 mM phosphate (8). In normal rats, serum calcium concentrations are 1.25 to 1.30 mM. In vitro, these levels of calcium result in a rate of PTH secretion that is close to the maximal inhibition of PTH secretion; in vivo experiments in rats also demonstrated that PTH secretion with basal calcium concentrations is relatively close to maximal inhibition (22). This study was performed with parathyroid tissue slices, rather than dispersed cells, because previous studies demonstrated that the effect of phosphate on PTH secretion was observed only in tissue preparations and not in dispersed parathyroid cells in culture (11).

The addition of ionophore or thapsigargin to parathyroid tissue incubated with low calcium levels and normal phosphate levels reduced PTH secretion to the level observed with 1.35 mM calcium. Inhibition of PTH secretion with the addition of ionophore has been described by other authors (23,24). The use of thapsigargin in the medium also reduced PTH release, which is in agreement with previous work by others (18,25). Thapsigargin increases cytosolic calcium levels by stimulating the release of calcium from intracellular stores, a mechanism by which high extracellular calcium levels produce increases in intracellular calcium concentrations. CaR binding results in G protein-dependent activation of phosphatidylinositol-specific PLC, causing accumulation of inositol-1,4,5-triphosphate and 1,2-sn-diacylglycerol and promoting rapid release of calcium from intracellular stores (26); the high cytosolic calcium level is sustained by calcium influx across the cell membrane (2). Our findings of inhibition of PTH secretion by both the ionophore and thapsigargin confirm the important role of intracellular calcium as a mediator of the inhibition of PTH secretion by high extracellular calcium levels. The inhibition of PTH secretion with the elevation of cytosolic calcium levels was associated with an increase in AA production, suggesting that the elevation of intracellular calcium levels is involved in PLA2 activation. A recent study by Kifor et al. (6) suggested that, in bovine parathyroid cells, the CaR mediated activation of PLA2 through the MAP kinase cascade; in the same work, the activation of MAP kinases by high calcium levels was prevented by inhibition of PLC, suggesting that PLC-dependent activation of PKC and/or elevation of cytosolic calcium levels was responsible for the activation of MAP kinases. Our results demonstrate that an increase in cytosolic calcium levels activates PLA2, which supports the role of PLC in PLA2 activation in parathyroid cells.

High extracellular phosphate levels reduced the production of AA by parathyroid cells, despite a high calcium concentration (1.35 mM) in the medium (Figure 2); this is a mechanism by which high extracellular phosphate levels may stimulate PTH secretion (17). The addition of ionophore or thapsigargin to the high-calcium medium with a normal phosphate level did not further increase the production of AA. This result suggests that cytosolic calcium-dependent PLA2 activation is fully accomplished with 1.35 mM calcium, which is the calcium concentration that produced maximal inhibition of PTH secretion in our in vitro model. A key finding in our study was that an elevation of cytosolic calcium levels was able to significantly increase AA production, despite high phosphate levels in the medium, and to reduce PTH secretion to values similar to those observed with normal phosphate levels in the medium. These results support the hypothesis that the reduction in AA production induced by high extracellular phosphate levels is attributable to an inadequate increase in cytosolic calcium levels in response to stimulation of CaR by calcium.

With low calcium concentrations (0.6 mM), PLA2 seems to be inactive, because production of AA was depressed; an increase in extracellular phosphate levels did not further reduce AA production. The addition of ionophore or thapsigargin increased AA production to the same levels with normal and high phosphate levels, suggesting again that high cytosolic calcium concentrations overcome the effects of phosphate. Therefore, high phosphate levels do not affect the ability of active PLA2 to produce AA; rather, the defect in AA production is at earlier steps in the intracellular signaling pathway.

With a normal phosphate concentration, PTH secretion was reduced by a high calcium concentration, and the addition of compounds that elevate cytosolic calcium concentrations did not further reduce PTH secretion (Figure 3), suggesting that a maximal inhibitory signal was obtained with 1.35 mM calcium. In contrast, a high phosphate concentration stimulated PTH secretion despite a high calcium concentration in the medium. With the same high phosphate concentration, both the ionophore and thapsigargin produced marked decreases in PTH secretion, to values similar to those obtained with normal phosphate levels. Therefore, the increase in cytosolic calcium levels was able to prevent the decrease in AA production and the stimulation of PTH secretion induced by high phosphate levels. These data support the theory that high phosphate levels interfere with normal increases in cytosolic calcium levels in response to increases in extracellular calcium concentrations. The results obtained with low calcium levels in the medium were similar to those observed with high calcium concentrations (Figure 4). With low extracellular calcium levels, the increase in intracellular calcium levels reduced PTH secretion to the same degree with both normal and high phosphate concentrations.

In conclusion, our results demonstrate that, in parathyroid tissue, an elevation in cytosolic calcium levels stimulates the PLA2-AA pathway and prevents the inhibition of AA synthesis induced by high phosphate concentrations. The mechanism by which high phosphate levels stimulate PTH secretion despite high extracellular calcium levels may depend on low cytosolic calcium levels, which impair activation of the PLA2-AA pathway. Further studies will be required to investigate how high phosphate levels affect the regulation of intracellular calcium levels.


   Acknowledgments
 
This work was supported in part by grants from the Ministry of Health (PB99-0768) and Junta de Andalucía, SAS (JA99/190). During the course of this work, Dr. Canalejo was a research fellow (Beca de Perfeccionamiento, Grant 97/4336) from the Instituto de Salud Carlos III, Ministry of Health. Dr. Almadén was supported by Fundación Hospital Reina Sofía-Cajasur.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brown EM, Gamba G, Riccardi D, Lombardi D, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC: Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature (Lond) 366: 575–580, 1993[CrossRef][Medline]
  2. Brown EM: Extracellular Ca2+ sensing, regulation of parathyroid cell function and role of Ca2+ and other ions as extracellular (first) messengers. Physiol Rev 71: 371–411, 1991[Free Full Text]
  3. Bourdeau A, Souberbielle J-C, Bonnet P, Herviaux P, Sachs C, Lieberherr M: Phospholipase A2 action and arachidonic acid metabolism in calcium-mediated parathyroid hormone secretion. Endocrinology 130: 1339–1344, 1992[Abstract]
  4. Bourdeau A, Moutahir M, Souberbielle JC, Bonnet P, Herviaux P, Sachs C, Lieberherr M: Effects of lipoxygenase products of arachidonate metabolism on parathyroid hormone secretion. Endocrinology 135: 1109–1112, 1994[Abstract]
  5. Kifor O, Diaz R, Butters R, Brown EM: The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK 293) cells. J Bone Miner Res 12: 715–725, 1997[CrossRef][Medline]
  6. Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchy T, Yao T, Kifor I, Brown EM: Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR transfected HEK293 cells. Am J Physiol 280: F291–F302, 2001
  7. Bonventre JV: Phospholipase A2 and signal transduction. J Am Soc Nephrol 3: 128–150, 1992[Abstract]
  8. Almaden Y, Canalejo A, Hernandez A, Ballesteros E, Garcia-Navarro S, Torres A, Rodriguez M: Direct effect of phosphorus on parathyroid hormone secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 11: 970–976, 1996[Medline]
  9. Kilav R, Silver J, Naveh-Many T: Parathyroid hormone gene expression in hypophosphatemic rats. J Clin Invest 96: 327–333, 1995
  10. Slatopolsky E, Finch J, Denda M, Ritter C, Zhong M, Dusso A, Mac Donald P, Brown A: Phosphate (PO4) restriction prevents parathyroid cell growth in uremic rats and high phosphate directly stimulates PTH secretion in tissue culture. J Clin Invest 97: 2534–2540, 1996[Medline]
  11. Nielsen PK, Feldt-Rasmusen U, Olgaard K: A direct effect of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells. Nephrol Dial Transplant 11: 1762–1768, 1996[Abstract/Free Full Text]
  12. Hernandez A, Concepcion MT, Rodriguez M, Salido E, Torres A: High phosphate diet increases prepro PTH mRNA independent of calcium and calcitriol in normal rats. Kidney Int 50: 1872–1878, 1996[Medline]
  13. Almaden Y, Hernandez A, Torregrosa V, Canalejo A, Sabate L, Fernandez Cruz L, Campistol JM, Torres A, Rodriguez M: High phosphate directly stimulates PTH secretion and synthesis by human parathyroid tissue. J Am Soc Nephrol 9: 1845–1852, 1998[Abstract]
  14. de Francisco ALM, Cobo MA, Setien MA, Rodrigo E, Fresnedo GF, Unzueta MT, Amado JA, Ruiz JC, Arias M, Rodriguez M: Effect of serum phosphate on parathyroid hormone secretion during hemodialysis. Kidney Int 54: 2140–2145, 1998[CrossRef][Medline]
  15. Moallem E, Kilav R, Silver J, Naveh-Many T: RNA-protein binding and post-transcriptional regulation of parathyroid hormone gene expression by calcium and phosphate. J Biol Chem 273: 5253–5259, 1998[Abstract/Free Full Text]
  16. Rodriguez M, Almaden Y, Hernandez A, Torres A: Effect of phosphate on the parathyroid gland: Direct and indirect? Curr Opin Nephrol Hypertens 5: 321–328, 1996[CrossRef][Medline]
  17. Almaden Y, Canalejo A, Ballesteros E, Añon G, Rodriguez M: The effect of high extracellular phosphate concentration on arachidonic acid production by parathyroid tissue in vitro. J Am Soc Nephrol 11: 1712–1718, 2000[Abstract/Free Full Text]
  18. Thastrup O, Cullemn PJ, Drobak BK, Hanley MR, Dawson AA: Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87: 2466–2470, 1990[Abstract/Free Full Text]
  19. Breitwieser GE, Gama L: Calcium-sensing receptor activation induces intracellular calcium oscillations. Am J Physiol 280: C1412–C1421, 2001[Abstract/Free Full Text]
  20. Cardenas S, Ballesteros E, Gallego M, Valcarcel M: Sequential determination of triglycerides and free fatty acids in biological fluids by use of continuous pretreatment module coupled to gas chromatograph. Anal Biochem 222: 332–341, 1994[CrossRef][Medline]
  21. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976[CrossRef][Medline]
  22. Lewin E, Almaden Y, Rodriguez M, Olgaard K: PTHrP enhances the secretory response of PTH to a hypocalcemic stimulus in rat parathyroid glands. Kidney Int 58: 71–81, 2000[CrossRef][Medline]
  23. Habener JF, Stevens TD, Ravazzola M, Orci L, Potts JT: Effects of calcium ionophores on the synthesis and release of parathyroid hormone. Endocrinology 101: 1524–1537, 1977[Medline]
  24. Mihai R, Lai T, Schofield GJ, Farndon JR: Changes in cytoplasmic calcium determine the secretory response to extracellular cations in human parathyroid cells: A confocal microscopy study using FMI-43 dye. Biochem J 352: 353–361, 2000
  25. Shoback D, Chen TH, Pratt S, Lattyak B: Thapsigargin stimulates intracellular calcium mobilization and inhibits parathyroid hormone release. J Bone Miner Res 19: 743–750, 1995
  26. Brown EM, Vassilev PM, Quinn S, Hebert SC: G-protein-coupled, extracellular Ca2+-sensing receptor: A versatile regulator of diverse cellular functions. Vitam Horm 55: 1–71, 1999[Medline]
Received for publication April 20, 2001. Accepted for publication September 6, 2001.




This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
T. Drueke, D. Martin, and M. Rodriguez
Can calcimimetics inhibit parathyroid hyperplasia? Evidence from preclinical studies
Nephrol. Dial. Transplant., July 1, 2007; 22(7): 1828 - 1839.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. Rodriguez, E. Nemeth, and D. Martin
The calcium-sensing receptor: a key factor in the pathogenesis of secondary hyperparathyroidism
Am J Physiol Renal Physiol, February 1, 2005; 288(2): F253 - F264.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
J. Silver, R. Kilav, and T. Naveh-Many
Mechanisms of secondary hyperparathyroidism
Am J Physiol Renal Physiol, September 1, 2002; 283(3): F367 - F376.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Almadén, Y.
Right arrow Articles by Rodríguez, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Almadén, Y.
Right arrow Articles by Rodríguez, M.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP