ARTICLE |
Correspondence to: Marco R. Celio, Inst. of Histology and General Embryology, U. of Fribourg, Fribourg, Switzerland. E-mail: marco.celio@unifr.ch
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Summary |
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The parathyroid glands are of major importance in calcium homeostasis. Small changes in the plasma calcium (Ca2+) concentration induce rapid changes in parathyroid hormone (PTH) secretion to maintain the extracellular Ca2+ levels within the physiological range. Extracellular Ca2+ concentration is continuously measured by a G-protein-coupled Ca2+-sensing receptor, which influences the expression and secretion of PTH. The mechanism of signal transduction from receptor sensing to PTH secretion is not well understood, but changes in PTH secretion are tightly linked to changes in the cytosolic Ca2+ concentration. Using immunohistochemistry and Western blot analysis, we detected the EF Ca2+ binding protein parvalbumin (PV) in normal and in hyperplastic and adenomatous human parathyroid glands. The strongest PV signal was present in chief cells and water clear cells, whereas in oxyphilic cells only a weak signal was observed. Immunohistochemistry and in situ hybridization of the PTH indicated a co-localization of PV and PTH in the same cell types. Because changes in the cytosolic Ca2+ concentration are believed to influence the process of PTH secretion, a possible role of PV as a modulator of this Ca2+ signaling is envisaged. (J Histochem Cytochem 48:105111, 2000)
Key Words: calcium buffering, calcium homeostasis, EF-hand, parathyroid, parathyroid hormone, parvalbumin
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Introduction |
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Calcium (Ca2+) is implicated in the regulation of a variety of cellular processes, such as muscle contraction, cell division, cell fertilization, phagocytosis, hormone secretion, glucose metabolism, and even gene expression (
The important role of the parathyroid gland as an overall regulator of Ca2+ homeostasis has been related to its exquisite capacity to sense and respond to minimal variations in the extracellular Ca2+ concentration (
The intracellular Ca2+ concentration is also tightly regulated by the ATP-dependent Ca2+ pump and by different types of Ca2+ exchangers located at the plasma membrane, endoplasmic/sarcoplasmic reticulum, and mitochondria (
PV is found in lower and higher vertebrates, including humans ( and ß, can be distinguished by their different biophysical characteristics (
-PV form is expressed (
In this study we looked for the presence of PV in parathyroid glands with different states of activity: normal, hyperplastic and adenomatous tissue. We report on the presence of PV in normal as well as diseased parathyroid glands and co-localization with PTH.
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Materials and Methods |
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Isolation of Tissue
Human parathyroid tissue was obtained from surgical procedures. Normal parathyroid glands (10 specimens: six men, four women, age range 2668) were isolated from thyroidectomy biopsies removed for goiter (four cases), follicular adenoma (five cases), and papillary carcinoma (one case). All eight cases of secondary hyperplasia (five men, three women, age range 2969) were related to chronic renal failure. Ten specimens of dissected parathyroid adenoma (four men, six women, age range 4676) were received fresh from the Department of Pathology of the Ospedale Civile di Belluno, Italy.
Immunohistochemistry
Tissues were routinely fixed in 10% neutral buffered formalin and embedded in paraffin. Immunohistochemical reactions were carried out according to the avidinbiotinylated peroxidase complex method (ABC) (Vector Laboratories; Burlingame, CA) as previously reported (
In Situ Hybridization
Dewaxed and rehydrated tissue sections were digested using proteinase K (Sigma). Fluorescein-labeled parathormone oligonucleotide probe (Novocastra; New Castle, UK) was added to each section. A rabbit F(ab') anti-FITC antiserum conjugated with alkaline phosphatase (Novocastra) was subsequently applied and the reaction developed with nitroblue tetrazolium (NTB).
Tissue Extraction and Western Blot Analysis
Frozen tissue was extracted by sonication with 5 volumes of 4 mM EDTA, pH 7.0, supplemented with 1 µM pepstatin hemisulfate A, 0.4 mM phenylmethylsuphonyl fluoride, 150 µM L-1-(tosylamido)-2-phenylethyl chloromethyl ketone, 1 µM leupeptin, and 30 U/ml trasylol. Protein concentration of cleared supernatants was measured using the microbiuret assay (
Tissue extracts (60 µg/lane) were separated by 15% SDS-PAGE. Separated lysates were transferred onto nitrocellulose membrane (Hybond ECL; Amersham Life Science, Poole, UK). Membranes were blocked in 3% bovine serum albumin, 1% fetal calf serum in 200 mM NaCl, 1 mM CaCl2, and 50 mM Tris-HCl, pH 7.4, and then incubated with primary polyclonal antisera diluted in blocking solution. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (Sigma). Staining of secondary antibody was performed using the ECL technique (Super Signal Substrate; Pierce, Rockville, IL).
Transblot 45Ca2+ Overlay
The protocol of
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Results |
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Immunohistochemistry with Antibodies Against Different EF-hand Ca2+ Binding Proteins
To investigate whether PV, calbindin D-28k, and calretinin are present in normal and pathological parathyroid glands, 20 samples were analyzed by immunohistochemistry. In all cases strong PV-immunoreactive staining was found, indicating the presence of PV in normal (Figure 1A) and hyperplastic (Figure 1B) parathyroid, as well as adenoma (Figure 1C) and carcinoma (Figure 1D) of parathyroid glands. The same localization of PV-immunoreactive staining was found for three different antibodies directed against PV from different species. No immunoreactive signal was observed using antibodies directed against calbindin D-28k and calretinin (not shown).
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PV immunoreactivity was localized in the chief cells (dark and light) and their morphological variants: the water clear cells and oxyphilic (oncocytic) cells (Figure 1A1E). However, the intensity and intracellular localization of the immunoreactive staining was very different among the various cell types. Strongest PV signals were found in chief cells, with an even distribution throughout the cytoplasm and a variable staining in the nucleus. In water clear cells, strong PV staining was found in the cytoplasm close to the inner side of the plasma membrane and in the nucleus (Figure 1D). In oxyphilic cells, PV immunoreactivity was faint or absent (Figure 1E).
No immunostaining for any of the three Ca2+ binding proteins was found in thyroid gland, including C-cells (not shown).
Western Blot Analysis with Antibodies Against PV
Western Blot analysis was performed to demonstrate that the signals found in immunohistochemistry with different antibodies against PV were specifically due to the presence of human PV. Because normal human parathyroid glands are very small and difficult to isolate from the surrounding thyroid tissue, protein extracts of parathyroid tissue from hyperplasia and adenoma were used for this analysis (Figure 2).
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A total of four hyperplastic parathyroids and six adenomas were investigated. In all protein extracts, the antibodies against rat PV detected a protein band with the same molecular weight (Mr of 12,000) as expected for human PV, indicating that the antibodies indeed detected human PV in immunohistochemistry (Figure 2). A strong signal for human PV was observed using the ECL technique, whereas only week signals were found using the less sensitive chloronaphthol staining.
Transblot 45Ca2+ Overlay
45Ca2+ overlay assays were performed to evaluate whether the human PV in the protein extracts from parathyroid bind Ca2+ ions. A single 45Ca2+ signal for a protein band with an Mr of 10,000 was observed whereas at the position where PV is located no 45Ca2+ signal was found. It is therefore assumed that PV is present at a rather low concentration in human parathyroid glands.
Immunohistochemistry with Antibodies Against Human PTH
Immunohistochemistry with antibodies against human PTH was performed to compare the localization of PV and PTH in different cells of the parathyroid glands.
For all tissues, the immunoreactivity for PV and PTH showed the same distribution and localization among the three different cell types. PTH immunoreactivity (Figure 1F) was strongest in chief and water clear cells, whereas it was absent in oxyphilic cells.
Western Blot Analysis with Antibodies Against Human PTH
Western blot analysis was performed to check whether the PTH-immunoreactive signal in immunohistochemistry is human PTH. The same protein extracts of hyperplastic and adenomatous parathyroid were used as in Western blots for PV identification.
The immunoreactive protein band reacting with antibodies directed against human PTH migrated at the correct molecular weight for human PTH (Mr 10,000), indicating that the antibodies indeed detected human PTH in immunohistochemistry (Figure 2C).
In Situ Hybridization of Parathyroid Tissue for PTH mRNA
PTH mRNA was found in all cells that showed positive immunoreactivity for PTH and PV, supporting the notion that these cells are actively producing PTH (Figure 1G).
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Discussion |
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Our results show for the first time the presence of the EF-hand Ca2+ binding protein PV in normal and hyperplastic parathyroid glands as well as in adenoma and carcinoma of the human parathyroid. Calbindin D-28k and calretinin, two other EF-hand proteins of the same superfamily, were absent in these glands. PV was found by immunohistochemistry mainly in chief cells and water clear cells, whereas oxyphilic cells have a much lower concentration. In particular, the distribution of PV reactivity in nucleus and cytoplasm in normal and pathalogical parathyroid is different. It is predominantly cytoplasmic in normal (Figure 1A) and hyperplastic (Figure 1B) parathyroid and predominantly nuclear in adenomas and carcinomas of parathyroid. This is partially due to the fact that the nuclear reaction in normal gland is masked in part by a strong coloration with hematoxylin. However, no connection between the nuclear reactivity and the normal course of the pathology can be drawn. In the adenomas (Figure 1C and Figure 1E), PV reactivity is also present (with a lower intensity compared to the adenoma) in the remaining suppressed gland.
Nuclear reactivity was also observed with other Ca2+ binding proteins (S-100, calretinin), but the biological implication is yet unknown. The PV signal in immunohistochemistry was further confirmed by Western blot analysis. The Western blot signal was strong using ECL visualization, whereas only faint signal was found using the conventional chloronaphthol method, indicating that the PV concentration in parathyroid tissue is rather low. Accordingly, 45Ca2+ transblot overlay assays with parathyroid extracts did not identify a Ca2+ binding protein at the molecular range of PV. Because different dilutions of PV were tested and the detection limit for 45Ca2+ was found to be below 1 µg (not shown), this indicated that the loaded samples (60 µg/lane) contained less than 1 µg of PV. Nevertheless, in all cases PV immunohistochemistry resulted in a clear and reproducible staining of parathyroid but not of thyroid cells. Therefore, PV immunohistochemistry could be used as a marker to identify and distinguish parathyroid from thyroid tissue in particular cases, such as when parathyroid gland has a follicular aspect.
In humans, the immunohistochemical localization of PV has been extensively used to map many areas of the brain (
PV is believed to play an important role in intracellular Ca2+ buffering and in Ca2+/Mg2+ exchange (
Finally, the presence of CaR is not limited only to cells involved in mineral ion metabolism but has also been found in brain (
In conclusion, the presence of the Ca2+ binding protein PV in PTH-expressing parathyroid cells raises interesting questions regarding its potential role as a Ca2+ buffer and/or as a modulator of Ca2+ signals within these cells.
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Acknowledgments |
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Supported by Swiss National Foundation 3100. 47291.96.
We thank Dr Merdol Ibrahim for help in editing figures and for critical reading of this manuscript.
Received for publication April 2, 1999; accepted August 10, 1999.
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