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Research Unit and Nephrology Service, Reina Sofia University Hospital,
Córdoba, Spain.
Department of Physical and Analytical Chemistry, Technical University
School at Linares, University of Jaén,
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By contrast, the early signal transduction mechanisms involved in the stimulation of PTH release by low extracellular calcium are increasingly understood. Extracellular calcium concentration modulates PTH secretion via a G-protein-coupled calcium-sensing receptor (13). This effector system includes the hydrolysis of membrane phospholipids by phospholipase C (PLC), phospholipase D (PLD), and phospholipase A2 (PLA2) to generate the appropriate intracellular signals (14). High extracellular calcium is coupled to the activation of PLA2 and the formation of arachidonic acid (AA), a potent inhibitor of PTH release, which acts via the 12- and 15-lipoxygenase pathway (15, 16).
In previous in vitro studies, we have shown that a high extracellular phosphate concentration prevents the normal inhibition of PTH secretion by calcium (4). Because AA seems to be an important mediator for the reduction of PTH release by calcium, additional experiments were performed to determine whether AA is involved in the regulation of PTH secretion by high phosphate. The results of these experiments showed that despite the presence of high phosphate concentration in the medium, the addition of exogenous AA to the medium restored the capacity of a high calcium concentration to inhibit PTH secretion. Although these results suggest that AA plays a role in the regulation of PTH secretion by phosphate, they do not show that phosphate directly affects AA production by parathyroid cells. The present study was designed to determine whether a high phosphate concentration has an effect on the PLA2-AA pathway in parathyroid cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dog parathyroid tissue was used when the experiments required a large amount of tissue. Dog parathyroid glands were obtained from euthanized mongrel dogs, 2 to 5 yr old and weighing 13 to 20 kg, donated by the Center for Animal Control. These dogs were healthy and ingested a standard chow diet containing 1.2% calcium, 0.9% phosphate and 1200 IU/kg vitamin D for a least 1 mo before the experiment. Euthanasia was achieved by the administration of 10 mg of intramuscular ketamine followed by 1 g of thiopental as an intravenous bolus. The time required to complete the parathyroidectomy in the dog was approximately 3 min.
Intact rat parathyroid glands or small (1 mm (3)) pieces of dog parathyroid tissue were placed in individual wells containing 2 ml of incubation media resting inside a nylon basket; the glands were maintained in a constant rocking and shaking motion (AOS-0, SBS Instruments SA, Badalona, Spain) at 37°C. Before and after each experiment, parathyroid cells were mechanically dispersed, and greater than 80% cell viability was confirmed by trypan blue exclusion.
Incubation Medium
The incubation medium was prepared using a distilled-deionized water
(Elgastat UHQ PS, Elga Ltd, High Wycombe, Bucks, England) and was buffered (pH
7.4) and contained in 125 mM NaCl, 5.9 mM KCl, 0.5 mM MgCl2, 0.4 to
2.0 mM CaCl2, Na-pyruvate 1, glutamine 4, glucose 12, Hepes 25 with
human insulin 0.1 IU/ml, bovine serum albumin 0.1%, penicillin g 100
IU/ml, and streptomycin 100 mg/ml. Phosphorus was added in the form of
NaH2PO4 and Na2HPO4 in 1:2
proportion to achieve the desired phosphorus concentration. The calcium
concentration in the medium was modified by adding CaCl2. The
target calcium concentration was confirmed by measurement using a selective
electrode (model 634; Ciba Corning, Essex, England); the ionized calcium was
measured 10 min before and immediately after completion of each 1-h incubation
period. The ionized calcium values did not change after incubation. All
chemical products were obtained from Sigma (St. Louis, MO).
Effect of High Phosphate on AA Production by the Parathyroid
Cell
In a previous report, we showed that PTH secretion was increased by a high
phosphate concentration and that this effect of phosphate was completely
reversed by the addition of AA (20 µM)
(4). In the present experiment,
the effect of phosphate on AA production by parathyroid cells was tested.
AA production was measured using gas chromatography (17). In the first experiment, the effect of high calcium concentration on AA production was evaluated. Dog parathyroid tissue was incubated for 1 h each in a low (0.6 mM) and high (1.35 mM) calcium concentration. A second experiment, in which dog parathyroid tissue was incubated in normal (1 mM) and high (4 mM) phosphate concentration, was designed to evaluate the effect of a high phosphate concentration on AA production. 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 EDTA, and 0.5 mM DTT. The AA was quantified by gas chromatography as described elsewhere (17) (model 5890-A; Hewlett Packard, Avondale, PA); with the use of this method, the coefficient of variation for AA quantification was 2.5%. The protein content of the tissue sample was determined using the Bradford method (18).
To confirm that the effect of phosphate was also observed in incubations of dog parathyroid tissue, separate experiments were performed using dog parathyroid tissue in which hourly PTH production was measured in 1.35 mM calcium in the presence of a normal and high phosphate concentration. Intact PTH in the incubation media was measured using a human intact PTH IRMA kit (Nichols Institute, San Juan de Capistrano, CA). The intra- and interassay coefficients of variation were 4.3 and 4.7%, respectively. The DNA content of the tissue sample was measured by spectrophotometry after isolation using a kit (Purogene; Gentra Systems, Inc., Minneapolis, MN).
Effect of High Phosphate on AA Production by Glomerulosa Cells
To determine whether the effect of phosphate on AA production in
parathyroid cells was tissue specific, separate experiments were performed in
rat adrenal glomerulosa cells, which are known to increase AA production in
response to angiotensin II (AII)
(19). Rat adrenal tissue was
sliced into 1-mm (3) pieces and
incubated in the previously described medium with 1.35 mM calcium and either a
normal (1 mM) or a high (4 mM) phosphate concentration. AII (1 nM) or vehicle
was added to the medium, and after 120 min the tissue was homogenized for the
measurement of AA and protein content using the same methodology as described
for parathyroid tissue.
Effect of High Phosphate on the Inhibition of PTH Secretion by
Exogenous PLA2
Intact rat parathyroid glands were incubated with PLA2 in
concentrations of 0 (vehicle), 5, 25, and 50 mU/ml in the presence of normal
(1 mM) and high (4 mM) phosphate. The calcium concentration in the medium was
maintained at 0.6 mM; this low calcium concentration has been associated with
low endogenous PLA2 activity
(15). PTH secretion was
measured in the medium after a 1-h incubation period. The intact PTH
concentration in the incubation medium was measured using a rat intact PTH
IRMA kit (Nichols Institute). The intra- and interassay coefficients of
variation were 4.3 and 4.7%, respectively.
Effect of High Phosphate on Parathyroid Cell Production of AA
Stimulated by Exogenous PLA2
Dog parathyroid tissue was incubated in 1.35 mM and 0.6 mM calcium with 1
mM phosphate and either PLA2 (25 mU/ml) or vehicle added to the
medium. The experiments were repeated using a high (4 mM) phosphate in the
medium. The production of AA and protein content was determined in homogenized
tissue as described previously.
Effect of Sulfate on PTH Secretion
These experiments were performed to determine whether sulfate, an ion with
chemical characteristics similar to phosphate, has an effect on PTH secretion.
The effect of sulfate on PTH secretion was tested in vitro using
intact rat parathyroid glands. The experiments were performed following the
same methodology used in our previous study in which a direct effect of
phosphate on PTH secretion was shown
(4). Rat parathyroid glands (10
per well) were incubated for 9 h in the above described media using a calcium
and phosphate concentration of 1.25 and 1 mM, respectively. The incubation was
prolonged to 10 h because in previous studies
(4), the maximum effect of
phosphate on PTH secretion was observed between 5 and 10 h and other authors
have shown that the effect of phosphate on PTH secretion increases with time
(6). The concentration of
sulfate in the medium was either 0.5 or 4 mM. Adequate amounts of sodium
sulfate were added to the medium to obtain the desired concentration of
sulfate in the medium. During the tenth hour, the calcium concentration was
decreased to 0.6 mM to stimulate PTH secretion and the sulfate and phosphate
concentrations were unchanged. The PTH secretion rate was measured during both
the ninth and tenth hours of incubation.
Statistical Analyses
Differences between more than two means were evaluated by analysis of
variance followed by the Duncan test. Paired or unpaired t tests were
used to compare two means from the same or from different groups of glands,
respectively. The results are expressed as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In separate experiments using rat parathyroid glands, the addition of AA (20 µM) to a medium with normal (1 mM) phosphate and low (0.6 mM) calcium concentration resulted in a reduction of PTH secretion from 81 ± 7 to 32 ± 4 pg/µg DNA (P < 0.01). This low level of PTH secretion induced by AA was similar to that obtained with high (1.35 mM) calcium in the medium (34 ± 3 pg/µg DNA). The inhibition of PTH secretion by calcium was not further enhanced by the addition of AA.
In a high calcium (1.35 mM) concentration, an increase in phosphate concentration from 1 to 4 mM resulted in an increase in PTH secretion (34 ± 3 versus 86 ± 7 pg/µg DNA, P < 0.01); the addition of AA (20 µM) to a high phosphate medium reduced the PTH level to that obtained with normal (1 mM) phosphate concentration (39 ± 9 pg/µg DNA).
Effect of High Phosphate on AA Production by Parathyroid Cells
The time effect of normal (1 mM) and high (4 mM) phosphate concentration on
AA production by parathyroid tissue is shown in
Figure 1. In a medium with
normal phosphate concentration (1 mM), a calcium of 1.35 mM resulted in a
threefold and significant increase in AA production; the values subsequently
decreased to basal levels at 60 min. By contrast, in 4 mM phosphate and the
same high calcium concentration, the production of AA remained unchanged
during the 1-h observation period.
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The secretion of PTH by dog parathyroid tissue was determined during 3 h of incubation in 1.35 mM calcium with 1 or 4 mM phosphate. In 1 mM phosphate, PTH secretion remained unchanged (64 ± 5, 60 ± 6, and 61 ± 4 pg/µg DNA at 1, 2, and 3 h, respectively); by contrast, in 4 mM phosphate, PTH secretion increased progressively (76 ± 7, 82 ± 4, and 87 ± 5, pg/µg DNA at 1, 2, and 3 h, respectively). Values at 2 and 3 h were significantly increased (P < 0.05) in 4 mM phosphate as compared with 1 mM phosphate.
Effect of High Phosphate on AA Production by Adrenal Glomerulosa
Cells
To evaluate whether the effect of phosphate on AA production was specific
for parathyroid cells, separate experiments were performed in rat adrenal
glomerulosa tissue, which are known to increase AA production in response to
AII. The addition of AII (1 nM) to the medium containing 1 mM phosphate
induced a threefold increase in AA production (P < 0.05). With
high phosphate (4 mM) in the medium, the basal and AII-induced AA production
were not significantly different from those obtained with 1 mM phosphate in
the medium (Figure 2). Thus, in
glomerulosa cells, basal and AII-stimulated AA production were unaffected by
the extracellular phosphate concentration.
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Effect of High Phosphate on the Inhibition of PTH Secretion by
Exogenous PLA2
The effect of phosphate on the inhibition of PTH secretion in the presence
of exogenous PLA2 is shown in
Figure 3. With normal phosphate
(1 mM) in the medium and a low calcium (0.6 mM) concentration, the addition of
increasing concentrations of PLA2 inhibited PTH secretion in a
dose-dependent manner; the maximum inhibition of PTH secretion was achieved
with 25 mU/ml PLA2. By contrast, in 4 mM phosphate, the same
concentration of PLA2 did not reduce PTH secretion. Thus, exogenous
PLA2 in concentrations that induced maximum inhibition of PTH
secretion did not reduce the increased PTH secretion induced by high
phosphate.
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Effect of PLA2 on AA Production by Parathyroid Cells with
High Extracellular Phosphate
In a medium containing 1.35 mM calcium, the addition of PLA2 did
not further increase production of AA. An elevation of phosphate in the medium
from 1 to 4 mM resulted in a significant decrease in AA production
(Figure 4A), and the addition
of PLA2 (25 mU/ml) to the medium containing high phosphate did not
increase AA production (Figure
4A).
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With a low calcium concentration in the medium (0.6 mM), the AA production was low; however, in the presence of normal phosphate, the addition of PLA2 produced a significant increase in AA production. With low calcium and a high phosphate concentration in the medium, the AA production was low and was not stimulated by the addition of PLA2 to the medium (Figure 4B).
Effect of Sulfate on PTH Secretion
The modification of sulfate concentration in the medium did not
significantly change PTH secretion (Figure
5). With low calcium, the PTH secretion increased significantly
and the presence of a high sulfate concentration did not affect the
stimulation of PTH secretion by low calcium. Thus, in vitro, PTH
secretion was not affected by high sulfate concentration in the medium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our experiments show that in parathyroid cells, AA production is stimulated by high extracellular calcium and in the presence of a low calcium concentration, the addition of AA inhibits the PTH secretion. Similar results have been reported by others (14,15,16). The high calcium-induced AA production was observed after a 30- to 45-min incubation, and at 60 min the AA values had returned to baseline. This temporal effect of a high calcium concentration-induced AA production was also reported by Bourdeau et al. (15), but the peak production of AA was observed after 5 min. Such differences in the time effect were probably because these experiments were performed in dispersed parathyroid cells, whereas in our experiments, slices of parathyroid tissue were used. Kifor et al. (14) found that a high calcium (3 mM) concentration induced a progressive increase in AA until 30 min, but there was no further follow-up, so it was not possible to determine a peak effect. Nevertheless, these authors reported that AA production was increased in parathyroid cells exposed to high calcium concentration in the medium. Thus, the activation of PLA2-AA pathway likely functioned to mediate the inhibition of PTH secretion by calcium. The addition of high phosphate concentration to the medium produced a remarkable decrease in the AA production induced by high calcium (Figure 1), and the supplementation of AA prevented the stimulation of PTH secretion by high phosphate (4). These data suggest that high phosphate stimulates PTH secretion by inhibiting AA production.
To investigate whether the phosphate effect on PTH secretion was tissue specific, we performed additional experiments to evaluate the effect of high phosphate in adrenal glomerulosa tissue. Aldosterone production by adrenal glomerulosa cells is stimulated by AII, and this effect is mediated by activation of PLA2, which results in high AA production (19). Our results confirmed that AII stimulates AA production by adrenal glomerulosa cells; in fact, we observed a threefold increase in AA production, which is comparable to the twofold elevation in AA levels reported by Kojima et al. (19). A high phosphate concentration in the medium did not modify the basal AA or the AII-stimulated AA levels. These results suggest that the effect of phosphate is specific for parathyroid tissue. It has been proposed that the recently cloned PiT-1 Na/P cotransporter expressed in parathyroid cells may act as phosphate sensor. To our knowledge, this cotransporter is not expressed in adrenal glomerulosa cells and this may explain the lack of an effect of phosphate in these cells.
In parathyroid tissue incubated in a normal phosphate and low calcium concentration, the addition of PLA2 produced a decrease in PTH secretion. This effect of PLA2 was dose dependent, similar to the observation reported by Bourdeau et al. (15). The reduction of PTH secretion by PLA2 was associated with an increase in AA production. The addition of PLA2 to a medium with high calcium did not further increase the already elevated production of AA, suggesting that AA production was maximally stimulated by the high calcium. A high phosphate concentration in the medium prevented both effects of PLA2, the inhibition of PTH secretion and the increase in AA production by parathyroid tissue. The lack of response to PLA2 may suggest inability of phospholipids to release AA under the action of PLA2 in the presence of a high phosphate concentration.
The sensing mechanism for phosphate is unknown, but the newly described phosphate cotransporter is being proposed as a possible phosphate sensor (11, 12). Although there is not information about the intracellular events that mediate the regulation of PTH secretion by phosphate, it has been shown that three phospholipases, PLC, PLD, and PLA2, participate in parathyroid cell signaling (14). The results of the present study suggest that a high phosphate concentration regulates PTH secretion through the PLA2-AA signaling system. A role for PLC or PLD cannot be excluded, although our preliminary experiments indicate that inhibition of PLC-dependent protein kinase C activity does not modify the stimulatory effect of phosphate on PTH secretion. In rat parathyroid glands cultured in a low (0.6 mM) calcium and a normal phosphate (1 mM) concentration, the addition of H7 (10 µM), a protein kinase C inhibitor, reduced the PTH secretion from 75 ± 6 to 38 ± 7 pg/µg DNA, P < 0.01; however, with a high phosphate (4 mM) concentration, the addition of H7 did not decrease the PTH secretion significantly (89 ± 8 versus 87 ± 10 pg/µg DNA). These results suggest that intracellular signaling system for low calcium- and high phosphate-induced PTH stimulation are not the same.
Different groups have shown that the effect of phosphate on PTH secretion is observed only in tissue preparations rather than in dispersed parathyroid cells in culture. Nielsen et al. (7) performed studies to test whether the effect of phosphate on PTH secretion was influenced by the type of in vitro culture: dispersed bovine parathyroid cells versus slices of bovine parathyroid tissue. They showed that the stimulation of PTH secretion by phosphate was observed only in parathyroid tissue, but both dispersed cells and tissue preparations responded to changes in the calcium concentration. Slatopolsky et al. (20) presented data indicating that phosphate did not stimulate PTH secretion in dispersed bovine parathyroid cells. However, the same group of investigators, using intact rat parathyroid glands in culture, were able to show a direct effect of phosphate on PTH secretion (6). In a recent report by Roussanne et al. (21), an adequate PTH response to calcium and persistence of the calcium-sensing receptor was observed in a long-term human parathyroid cell culture. In these cells, a high phosphate concentration significantly increased PTH secretion. According to the authors, the observation of an effect of phosphate may have been possible as a result of the presence of clusters with close cell-to-cell interaction. Therefore, there is experimental evidence that shows that the effect of phosphate on parathyroid function is observed in intact tissue rather than dispersed cells in culture. However, there is not a clear explanation of why cell-to-cell interaction is important to observe an effect of phosphate on PTH secretion. Sun et al. (22) demonstrated that parathyroid cells in close proximity are stimulated to secrete more PTH and suggest the presence of a paracrine interaction among parathyroid cells. Following this same line of reasoning, intercellular communication may be required to observe an effect of phosphate on PTH secretion. The present work shows that a high phosphate concentration affects the production of AA by parathyroid cells. Thus, as in other systems (23), it is possible that eicosanoid products produced by a cell may affect neighbor cells. This may be the reason whereby intact parathyroid tissue architecture or at least a close cell-to-cell interaction is required to see an effect of phosphate.
As in our previous study (9), the results of the present study were obtained using normal parathyroid tissue. Hyperplastic parathyroid glands from humans or from uremic animals might not show the same response as normal parathyroid glands.
The rationale to evaluate a possible effect of sulfate on PTH secretion was simply that sulfate, like phosphate, is another divalent anion circulating in relatively high concentrations in the blood. A sulfate concentration as high as 4 mM did not have an effect on PTH secretion. This indicates that the effect of phosphate is not only tissue specific (parathyroid gland) but also specific for the phosphate ion.
In conclusion, a high phosphate concentration affects the production of AA by parathyroid tissue. This effect of phosphate may be the mechanism by which a high phosphate stimulates PTH secretion.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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