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Department of Internal Medicine 3, Kumamoto University School of Medicine, 1–1-1 Honjo, Kumamoto 860-8556, Japan
Address all correspondence and requests for reprints to: Hiroshi Tokunaga, M.D., Department of Internal Medicine 3, Kumamoto University School of Medicine, 1–1-1 Honjo, Kumamoto 860-8556, Japan. E-mail: tokunaga{at}kaiju.medic.kumamoto-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The parathyroid is a conditionally renewing tissue with very low turnover (2, 3), but it has the potential to proliferate under appropriate physiological or pathological stimuli such as hypocalcemia, high serum phosphate, or low levels of 1,25-dihydroxyvitamin D (4, 5), which are frequently observed in chronic renal failure. Bovine PT cells in primary culture were reported to proliferate under conditions of hypocalcemia, with a rapid, but transient, rise in c-Myc and c-Fos (6). It has also been reported that PT cell proliferation is derived from such genomic alterations as in cyclin D1/PRAD1 expression (7), menin (8), and RET protooncogene (9). However, it is still unclear exactly what factors are involved in PT cell proliferation (10).
Endothelin-1 (ET-1) was originally isolated in 1988 as a secretory product of cultured porcine aortic endothelial cells and was shown to be a potent vasoconstrictor and presser peptide (11). Subsequently, it has become clear that ET-1 exerts multiple biological effects on hormonal cells, including inhibition of renin secretion; stimulation of catecholamine; secretion of vasopressin and aldosterone; stimulation of LH, FSH, GH, and TSH secretion; inhibition of PRL release; as well as very strong vasoconstriction (12, 13). It has also been reported that ET-1 is a mitogenic factor functioning in an autocrine/paracrine manner in a number of different cell lines, including rat vascular smooth muscle cells (14), rat glomerular mesangial cells (15), Swiss 3T3 fibroblasts (16), and human cancer cells (17). The expression of ET receptors (ETA and ETB) has been reported in human and bovine parathyroid glands (18, 19), and that of ETA has been reported in rat PT cell lines (19). It was also reported that rat PT cells synthesize ET-1 and bear ET-1 receptors, which suggests an autocrine/paracrine role for ET-1 in PT cells (20). Along these evidentiary lines, we hypothesize that ET-1 may play an important role in the proliferation of PT cells and that ET receptor antagonist may have therapeutic benefit for secondary hyperparathyroidism as it reportedly has for heart failure (21), myocardial infarction (22), pulmonary hypertension (23), and renal disease (24). In our judgment, an in vivo study is more appropriate than an in vitro or ex vivo study for testing this hypothesis because there is a down-regulation of calcium-sensing receptor (CaR) in bovine PT cells in primary culture (25). Furthermore, an in vitro study entails the loss of another important feature of PT cells, i.e. loss of the ability to secrete PTH in rat PT cell lines (26).
The present studies were conducted to characterize the pathophysiological role of the ET system in the proliferation of PT cells in vivo using rats fed a low calcium (Ca) diet for 8 weeks as an animal model for secondary hyperparathyroidism. In this study we demonstrated that an ETA/ETB receptor antagonist, bosentan, prevented the PT cell proliferation and lowered the serum PTH levels stimulated by the low Ca diet, which indicated that ET-1 was one of the factors involved in PT cell proliferation. This result implies that an ET antagonist could be used in the treatment of secondary hyperparathyroidism.
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Materials and Methods |
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Animals
Male Sprague Dawley rats (CLEA Japan) at 8 weeks of age were
used for this study. Rats were killed in accordance with the ethical
standards of the institutional review committee.
Measurement of serum PTH, serum biochemistry, and plasma ET-1
Blood samples were taken from the rats for the histological
examination. PTH in rat serum was measured by a rat kit (IRA,
Immunotopics, Inc., San Clemente, CA). Serum biochemistry, including
total Ca, phosphate (P), sodium (Na), potassium (K), and chloride (Cl),
was performed with an autoanalyzer. Plasma ET-1 was measured using an
ET enzyme immunoassay kit (Immuno-Biological Laboratories Ltd., Gunma,
Japan) after plasma extraction by passage through
C18 Sep-Pak cartridges as described previously
(28).
Animal models with secondary hyperparathyroidism
The rats were divided into two groups, fed either the control
chow (Ca, 0.6%; P, 0.3%) or the low Ca chow (Ca, 0.003%; P, 0.3%),
and maintained for up to 8 weeks (29). The rats were
killed after the 8 weeks of diet treatment. A pair of parathyroid
glands was excised and weighed to ascertain the wet weight per body
weight. For the histological examination, thyroparathyroidectomized
tissues were used, and staining for proliferating cell nuclear antigen
(PCNA) (30) and ET-1 was performed.
Assessment of chronic effects of bosentan on parathyroid
proliferation
The rats were divided into four groups and maintained for up to
8 weeks as follows: 1) control chow (Ca, 0.6%; P, 0.3%) with no
bosentan; 2) control chow with bosentan (100 mg/kg·day); 3) low Ca
chow (Ca, 0.003%; P, 0.3%) with no bosentan, and 4) low Ca chow with
bosentan (100 mg/kg·day). After 8 weeks of treatment, the rats were
killed. The wet weight of a pair of parathyroid glands was weighed, and
PCNA and ET-1 staining was performed. Histological analysis of femoral
bone was performed at the end of the treatment.
Microscopic analysis
The parathyroid glands were fixed in 4% buffered formalin for
12–24 h for PCNA staining or in Bouin’s solution for 12–24 h for
ET-1 staining, embedded in paraffin, and cut into 4-µm-thick
sections. The sections were deparaffinized and incubated in 1%
H2O2/methanol for 20 min to
block endogenous peroxidase.
For PCNA staining the sections were placed in citrate buffer (pH 6.0) and kept at 95 C for 10 min to reveal masked antigen (5). After being treated with normal rabbit serum to prevent background staining, sections were incubated overnight at 4 C with mouse monoclonal anti-PCNA antibody (DAKO Corp., Glostrup, Denmark) at a final dilution of 1:400, followed by incubation for 30 min with rabbit antimouse Ig/horseradish peroxidase (DAKO Corp.). Color developments were achieved by incubation with diaminobenzidine tetrahydrochloride solution. Counterstaining was performed in hematoxylin. The numbers of PCNA-positive PT cells per total PT cells were counted in the parathyroid section with the largest gland area.
For ET-1 staining the sections were incubated overnight at 4 C with rabbit antiserum to ET-1 (Peninsula Laboratories, Inc., San Carlos, CA) at a final dilution of 1:400 and processed further using the avidin-biotin-complex peroxidase method (Vector Laboratories, Inc., Burlingame, CA). The slides were counterstained with hematoxylin.
The femoral bones were fixed in 4% buffered formalin for 12–24 h, decalcified with 5% trichloroacetic acid, embedded in paraffin, and cut into 4-µm-thick sections. The sections were stained with hematoxylin and eosin or Mallory-Azan.
Statistics
The mean ± SEM are presented. ANOVA followed
by Bonferroni’s correction for multiple comparisons were used to
compare the results of each group of experiments. P <
0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows serum biochemistry, serum PTH
levels, and plasma ET-1 levels in rats fed either the normal diet or
the low Ca diet for 8 weeks. The serum total Ca levels were
significantly decreased in rats fed the low Ca diet compared with those
in rats fed the control diet. Serum phosphate (P) levels did not differ
significantly between rats fed the low Ca diet and those fed the
control diet. Serum PTH levels were markedly elevated in rats fed the
low Ca diet compared with those in rats fed the control diet (8.8
± 2.5 vs. 1142.7 ± 127.0 pg/ml; control diet
vs. low Ca diet; mean ± SEM;
n = 6; P < 0.01). Plasma ET-1 levels did not
differ significantly between rats fed the low Ca diet and rats fed the
control diet. The wet weight of a pair of parathyroid glands per
100 g BW was much heavier in rats fed the low Ca diet than in rats
fed the control diet after 8 weeks of treatment (0.06 ± 0.01
vs. 2.05 ± 0.04 mg/100 g BW; control diet
vs. low Ca diet; mean ± SEM;
n = 4; P < 0.01).
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, a and b, shows PCNA
staining of thyroparathyroidectomy tissue in rats fed either the
control diet or the low Ca diet for 8 weeks. PCNA staining showed that
there were 8.2 PCNA-positive PT cells/1000 PT cells in the parathyroid
gland in rats fed the control diet, and there was an approximately
2.5-fold increase in this ratio in rats fed the low Ca diet for 8 weeks
(8.2 ± 0.8 vs. 19.8 ± 2.5 PCNA-positive PT
cells/1000 PT cells; control diet vs. low Ca diet; mean
± SEM; n = 6; P <
0.01).
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) than
in rats fed the control diet (Fig. 1c). These data indicated that ET-1
protein expression was increased in PT cells in rats with secondary
hyperparathyroidism.
Effect of the
ETA/ETB receptor
antagonist, bosentan, on parathyroid cells with secondary
hyperparathyroidism
To examine the effect of ET-1 in the parathyroid gland on the
progression of secondary hyperparathyroidism, we used the
ETA/ETB receptor
antagonist, bosentan, to block signaling from the ET receptor. The wet
weight of a pair of the parathyroid glands from rats fed the low Ca
diet was dramatically reduced by bosentan (1.60 ± 0.29
vs. 0.17 ± 0.13 mg/100 g BW; low Ca diet with no
bosentan vs. low Ca diet with bosentan; mean ±
SEM; n = 4; P < 0.01; Fig. 2). The wet weight of a pair of
parathyroid glands from rats fed the normal diet did not differ
significantly with bosentan (n = 4 and 5; Fig. 2). Figure 3, a and b, shows PCNA staining of
thyroparathyroidectomy tissue from rats fed the low Ca diet either with
no bosentan or with bosentan for 8 weeks. Bosentan significantly
reduced the number of the PCNA-positive PT cells in rats fed the low Ca
diet (14.3 ± 2.7 vs. 2.1 ± 1.3 PCNA-positive PT
cells/1000 PT cells; low Ca diet with no bosentan vs. low Ca
diet with bosentan; mean ± SEM; n = 6;
P < 0.01; Fig. 4). The
number of PCNA-positive PT cells did not differ significantly in rats
fed the normal diet with bosentan (Fig. 4). Bosentan also significantly
reduced serum PTH levels in rats fed the low Ca diet (Fig. 5). The total serum Ca levels did not
differ significantly in rats fed the low Ca diet with bosentan (Fig. 6). The immunohistochemical study showed
that bosentan had no effect on ET-1 protein expression in PT cells in
rats fed the low Ca diet (Fig. 3, c and d). These results lead us to
conclude that blocking ET-1 signaling during the course of secondary
hyperparathyroidism prevented the proliferation of PT cells with a
reduction of serum PTH levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we induced secondary hyperparathyroidism in rats
by giving them a low Ca diet for 8 weeks as previously reported
(4, 5). Serum total Ca levels were significantly reduced,
and serum PTH levels were markedly elevated in the low Ca diet group
compared with the control diet group. The number of PCNA-positive PT
cells in rats fed the low Ca diet was 2.5 times that in rats fed the
control diet after 8 weeks of treatment (Fig. 2). The ratio is
compatible with that previously reported by Naveh-Many et
al. (5), where rats receiving a low Ca diet for 10
days had a 3.6-fold increase in PCNA-positive PT cells compared with
control rats. It was reported that in secondary hyperparathyroidism,
hypocalcemia is the major regulator of PT cell proliferation (2, 5, 10). Gq protein-coupled cell surface
CaR (32) plays the central role in sensing extracellular
calcium ([Ca2+]e) and has an exquisite
sensitivity to changes in [Ca2+]e
(33). However, the exact mechanism of how decreases in
stimuli of CaR or low serum calcium levels lead to an increase in PT
cell proliferation is not fully understood. The in vitro
study with primary culture of bovine PT cells has shown that PT cell
proliferation was preceded by a rapid and transient rise in c-Myc and
c-Fos (6), but it has also been shown that bovine PT cells
in primary culture lose CaR (25), which led us to conclude
that an in vivo study was necessary in the case of PT
cells.
The particularly interesting finding of our study was that ET-1 expression was increased during the course of the proliferation of the parathyroid gland by the low Ca diet. It has been shown that basal ET-1 secretion is enhanced in response to a variety of stimuli, such as thrombin, angiotensin II, and ischemia/reperfusion in cultured porcine aortic endothelial cells (34), cultured bovine endothelial cells (35, 36), and isolated perfused rat hearts (37). ET-1 expression has also been reported to be affected by the changes in [Ca2+]e or intracellular calcium ([Ca2+]i). Basal ET-1 production was not affected by reducing [Ca2+]e in cultured bovine and porcine endothelial cells, whereas agonist-stimulated ET-1 release was diminished by reducing [Ca2+]e (36, 38). Basal secretion of ET-1 from cultured porcine aortic endothelial cells was reduced by either decreasing or increasing [Ca2+]i (39). ET-1 secretion was inversely regulated by [Ca2+]e in bovine parathyroid cells (40). A decrease in [Ca2+]e induced a concentration-dependent decrease in [Ca2+]i in the rat PT cell lines (19). Our results provided evidence that the long-term reduction of [Ca2+]e, perhaps followed by a reduction of [Ca2+]i, induces the increase in ET-1 protein in PT cells in vivo. However, there was no difference in plasma ET-1 levels between rats fed the control diet and rats fed the low Ca diet. ET-1 levels in plasma are reported to represent spillover from endothelial cells (41). Indeed, given the minuscule size of the parathyroid glands, we would not have expected any significant rise in the plasma level in any event, even though it appears certain that ET-1 is synthesized in PT cells to a considerable extent. The parathyroid gland, which accounted for only 0.002% of total body weight even in rats fed the low Ca diet, is simply too small for its cells’ ET-1 output to show up as elevated plasma ET-1 levels. Thus, we believe that ET-1 functions in an autocrine/paracrine manner, not in an endocrine manner.
ET-1 is shown to act as a mitogen on PT cells, because blocking the signal from the ET receptor by an ETA/ETB receptor antagonist, bosentan, prevents PT cell proliferation induced by the low Ca diet. ET-1 has been reported to act as a mitogen and to stimulate cell division in a variety of cell lines, such as rat vascular smooth muscle cells (14), rat glomerular mesangial cells (15), Swiss 3T3 fibroblasts (16), and human cancer cells (17). It has been shown that in some endocrine cells, such as thyroid cells or endometrial cells, ET-1 plays a role in both cell proliferation and regulation of hormone secretion (12). ET-1 binds ET receptors and induces the expression of c-Fos and c-Myc, which leads to cell proliferation (15). In addition, ET-1 stimulates mitogen-activated protein kinase (42). ET-1 proliferates cells through trans-activation of the epidermal growth factor receptor (43). ET-1 stimulates the proliferation of rat adrenal zona glomerulosa cells in an autocrine/paracrine manner, acting through its ETA receptors coupled with protein kinase C- and tyrosine kinase-dependent signaling pathways (44). The proliferation of PT cells might be involved in any of these mechanisms.
Human parathyroid adenoma and human parathyroid gland with hyperplasia, and bovine parathyroid gland possess both ETA and ETB receptors (18, 19). However, rat cloned PT cell lines express only ETA receptor messenger RNA (19). The expression of both ETA and ETB receptors in human and bovine parathyroid glands, and not of ETA receptor alone as in rat PT cells, may be due to the coexistence of the endothelial cells that abundantly express ETB receptor. It might be reasonable to speculate that the inhibition of PT cell proliferation by bosentan was mediated by blocking signaling from the ETA receptor. We used a particular ETA/ETB receptor antagonist, bosentan, for this study, because it has several advantages. First, because this chemical is nonpeptide resistant to the digestive enzymes (27), it can be administered orally. Secondly, it could be obtained in large enough quantity for a study of up to 8 weeks in rats. However, further separate studies with specific ETA antagonists or ETB antagonists will be necessary to test the validity of our speculation. It is unlikely that bosentan has any toxic effect on PT cells, because it had no effect on PT cells in rats fed the control diet.
Bosentan significantly reduced serum PTH levels in rats fed the low Ca diet, but had no effect on serum PTH levels in rats fed the control diet. However, the reduction of PTH secretion by bosentan was less striking when we compared it with the reduction of the wet weight of the parathyroid glands or PCNA-positive PT cell number in rats fed the low Ca diet with bosentan. The exact reason for this discrepancy in the ratio of the reduction is uncertain, but it can be explained by one recent report. In bovine PT cells in primary culture, ET-1 has direct inhibitory effects on PTH secretion in low (0.5 or 0.7 mM) [Ca2+]e, whereas ET-1 has no effect on PTH secretion in high (1.5 or 2 mM) [Ca2+]e (40). Under low [Ca2+]e conditions, such as those to which the rats fed the low Ca diet in our experiments were subjected, blocking of ET-1 signals by bosentan could stimulate PTH release from PT cells, which would blunt the reduction of PTH secretion caused by preventing the PT cell proliferation caused by bosentan. However, there is also a conflicting report about the effects of ET-1 on PTH release, in which ET-1 has stimulatory effects on PTH secretion with high (4.0 mM) to low (0.2 mM) [Ca2+]e in bovine PT cells in primary culture (19).
Bosentan did not affect serum total Ca levels or bone histology, although it reduced serum PTH levels in rats fed the low Ca diet. We think it is because the reduction of about one third in PTH levels with bosentan to about two thirds the levels of those with no bosentan was not enough to effect the changes in serum total Ca levels or bone histology in rats fed the low Ca diet. The PTH levels in rats fed a low Ca diet, even when lowered with bosentan, were extremely high, really a different order of magnitude, compared with those in rats fed the control diet in our experiments.
In conclusion, our study suggests that ET-1 in PT cells has a role in parathyroid cell proliferation induced by hypocalcemia in an autocrine/paracrine manner in vivo. We also showed that the ETA/ETB receptor antagonist, bosentan, inhibited PT cell proliferation stimulated by hypocalcemia, which indicated that an ETA/ETB receptor antagonist may be one of the candidate drugs to prevent secondary hyperparathyroidism.
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Acknowledgments |
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Received June 2, 2000.
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