Journal of Histochemistry and Cytochemistry, Vol. 48, 105-112, January 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Parvalbumin Is Expressed in Normal and Pathological Human Parathyroid Glands

Thomas L. Paulsa, Fiorella Portisa, Ettore Macrìc, Brigitte Belsera, Philipp Heitzb, Claudio Doglionic, and Marco R. Celioa
a Institute of Histology and General Embryology, University of Fribourg, Fribourg, Switzerland
b Institut für Pathologie, Universität Zürich, Zürich, Switzerland
c Servizio di Anatomia Patologica Ospedale Civile di Belluno, Belluno, Italy

Correspondence to: Marco R. Celio, Inst. of Histology and General Embryology, U. of Fribourg, Fribourg, Switzerland. E-mail: marco.celio@unifr.ch


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 48:105–111, 2000)

Key Words: calcium buffering, calcium homeostasis, EF-hand, parathyroid, parathyroid hormone, parvalbumin


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Bading et al. 1993 ; Peunova and Enikolopov 1993 ). Because Ca2+ regulates such a multitude of processes, the maintenance of a steady concentration of Ca2+ both intra- and extracellularly is tightly regulated by several feedback control systems: Ca2+ absorption in the intestine, reabsorption/excretion in the kidney, and deposition/solubilization in the bones (Brown 1991 ; Hurwitz 1996 ). These three systems are regulated by three different hormones: parathyroid hormone (PTH), calcitonin (CT), and 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3], respectively.

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 (Brown 1991 ). Two different Ca2+-sensing receptors have been identified and cloned from parathyroid cells (Brown et al. 1993 ; Garrett et al. 1995 ). One of these receptors (the calcium receptor, CaR) belongs to the seven-transmembrane-helix G-protein-coupled receptor superfamily. Ca2+-dependent CaR activation results in the intracellular accumulation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Brown 1991 ). An increase in intracellular Ca2+ on CaR stimulation has been described and linked to extracellular Ca2+-mediated alterations in PTH secretion (Brown 1991 ). A second, less-studied putative Ca2+ sensor (CAS) is a large protein (Mr 500,000) with a single transmembrane region, belonging to the low-density lipoprotein (LDL) receptor family (Lundgren et al. 1994 ).

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 (Pietrobon et al. 1990 ). In addition, levels of intracellular Ca2+ are modulated by cytosolic Ca2+ binding proteins, of which the superfamily of EF-hand Ca2+ binding proteins is the largest and best characterized (Kretsinger 1980 ; Kawasaki and Kretsinger 1994 ). The best-known proteins of this superfamily are calmodulin, troponin C, calbindin D-28k, and PV.

PV is found in lower and higher vertebrates, including humans (Wnuk et al. 1982 ; Pauls et al. 1996 ). Several PV isoforms exist, which have been placed in a subfamily of the superfamily of EF-hand Ca2+ binding proteins (Moncrief et al. 1990 ; Nakayama et al. 1992 ). Two different isoforms, {alpha} and ß, can be distinguished by their different biophysical characteristics (Fohr et al. 1993 ). However, in adult human only a single {alpha}-PV form is expressed (Fohr et al. 1993 ) whereas a ß-PV is found exclusively extraembryonically in the placenta and in human tumors (Heizmann and Berchtold 1987 ). PV has been localized in metabolically active cells such as fast-twitch muscle fibers, GABAergic neurons, and several endocrine glands (Berchtold 1989 ). PV can function as a cytosolic Ca2+ buffer, thereby acting as a fast relaxation factor in muscle contraction or protecting cells from cytotoxic Ca2+ overload (Pauls et al. 1996 ). A recent study on PV-knockout mice shows that PV may confer an advantage in the performance of rapid, phasic movements in fast-twitch muscle fibers (Schwaller et al. 1999 ). Furthermore, a possible indirect role of PV in signal transduction by modifying the Ca2+ signal has been proposed (Pauls et al. 1996 ).

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.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Isolation of Tissue
Human parathyroid tissue was obtained from surgical procedures. Normal parathyroid glands (10 specimens: six men, four women, age range 26–68) 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 29–69) were related to chronic renal failure. Ten specimens of dissected parathyroid adenoma (four men, six women, age range 46–76) 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 avidin–biotinylated peroxidase complex method (ABC) (Vector Laboratories; Burlingame, CA) as previously reported (Doglioni et al. 1987 ). Three different antibodies against PV were used: a polyclonal antiserum 4064 against rat muscle PV (SWant; Bellinzona, Switzerland), a monoclonal antibody PA-235 against carp muscle PV (Sigma; St Louis, MO), and a monoclonal antibody Parv-19 against frog muscle PV (Sigma). The antiserum 224-1 against human PTH was from Signet (Dedham, MA). Antisera against calbindin D-28k and calretinin were from SWant. Antibody dilutions were 1:4000 for PA-235, 1:2000 for Parv-19, 1:3000 for 224-1, and 1:10,000 for anti-calretinin. The specificity of these antibodies was extensively tested (Kagi et al. 1987 ; Celio et al. 1988 ). Immunoreactivity was semiquantitatively evaluated with regard to the percentage of immunostained cells.

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 (Itzhaki and Gill 1964 ).

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 Maruyama et al. 1984 was followed. Proteins were first subjected to SDS-PAGE, then transferred to nitrocellulose membrane and finally incubated with 45Ca2+ (specific activity 10–40 mCi/mg; Amersham). 45Ca2+ binding proteins were visualized by autoradiography.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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).



View larger version (147K):
[in this window]
[in a new window]
 
Figure 1. Immunohistochemical localization of PV and PTH in normal and pathological parathyroid glands. Sections of normal parathyroid (A), hyperplasia (B), adenoma water clear cell with marked PV nuclear reactivity (C), carcinoma (D), adenoma oxyphilic cell with PV faint reactivity (E) were immunostained with primary antibodies against rat PV (A–E) and human PTH (F). Sections were counterstained with hematoxylin and antibodies were visualized by the ABC method. (G) In situ hybridization with a PTH-probe. Original magnification x 200.

PV immunoreactivity was localized in the chief cells (dark and light) and their morphological variants: the water clear cells and oxyphilic (oncocytic) cells (Figure 1A–1E). 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).



View larger version (13K):
[in this window]
[in a new window]
 
Figure 2. Western blot analysis of human PV and human PTH. Proteins of soluble extracts from hyperplastic parathyroid and adenoma were separated by one-dimensional SDS-PAGE and stained with Coomassie brilliant blue (a) or transferred onto nitrocellulose membrane (b,c). PV- or PTH-immunoreactive protein bands were identified using antibodies against rat PV (b) or human PTH (c) and subsequent visualization by enhanced chemiluminescence. Rat PV (1 µg) (Lanes 1, 5), prestained low molecular weight protein standard (BioRad; Hercules, CA) (Lanes 2, 6, 10), protein extracts from hyperplasia (Lanes 3, 7, 11) and adenoma (Lanes 4, 8, 12), and human PTH (100 ng) (Lane 9).

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).


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Pauls et al. 1996 ). In addition, PV has been immunohistochemically detected in human muscle (Fohr et al. 1993 ) and Leydig cells (Davidoff et al. 1993 ). On Western blot analysis, the presence of PV was also reported in human cerebellum and kidney, and with the more sensitive RT-PCR method PV mRNA was detected in muscle spindles, thymus, lung, diaphragm, and heart (Fohr et al. 1993 ). The parathyroid glands mediate their endocrine function through the production of PTH (Brown 1991 ). The three different cell types are assigned to different activities in PTH expression. Whereas chief and water clear cells actively express high amounts of PTH, oxyphilic cells are functionally inactive (de Lellis 1993 ). Interestingly, alterations in PTH secretion caused by changes in extracellular Ca2+ concentrations are tightly linked to changes of intracellular Ca2+ levels (Brown 1991 ). A close inverse correlation has been found between PTH secretion and intracellular Ca2+ concentrations (Brown 1991 ). High extracellular Ca2+ causes a decrease in PTH secretion and elicits an initial spike of cytosolic Ca2+, which is followed by a sustained increase (Brown 1991 ) or by oscillatory elevations of the cytosolic Ca2+ concentration (Miki et al. 1995 ; Ridefelt et al. 1995 ). On the basis of these data, it has been proposed that temporal or spatial characteristics of changes in the intracellular Ca2+ concentration may play a mediatory role in the regulation of PTH secretion (Brown 1991 ).

PV is believed to play an important role in intracellular Ca2+ buffering and in Ca2+/Mg2+ exchange (Pauls et al. 1996 ). Most PVs contain two functional metal ion binding sites that can bind Ca2+ with high affinity (KCa = 107–109 M-1) and Mg2+ with moderate affinity (KMg = 103–105 M-1) in a competitive fashion (Pauls et al. 1996 ). Under resting conditions, these sites are both occupied by two Mg2+ ions. An increase in the cytosolic free Ca2+ level to 1 µM or higher results in dissociation of Mg2+ and in subsequent binding of Ca2+ to both sites. Accordingly, Ca2+ spikes induced by brief depolarization pulses (Chard et al. 1993 ; Dreessen et al. 1996 ), ionophore, or KCl (Muller et al. 1996 ) are reduced in neurons microinjected with or overexpressing PV, demonstrating that PV buffers Ca2+ in a cytoplasmic environment. Therefore, it has been hypothesized that in neurons PV may act as a Ca2+ buffer that protects cells from cytotoxic Ca2+ overload (Nitsch et al. 1989 ; Yoshida et al. 1993 ; Pauls et al. 1996 ). In the parathyroid glands, PV may influence PTH expression or secretion by modifying intracellular Ca2+ signals. The same calcium receptor (CaR) of parathyroid glands is found in other tissues involved in Ca2+ homeostasis, e.g., kidney (Riccardi et al. 1995 ), and in C-cells of the thyroid gland (Garrett et al. 1995 ). Elevations of extracellular Ca2+ in both C-cells and chief cells of the parathyroid induce an increase in intracellular Ca2+. However, the consequences of this cytosolic Ca2+ increase are opposed. In chief cells it inhibits PTH secretion and in C-cells it stimulates calcitonin secretion (Fried and Tashjian 1986 ). Therefore, despite the fact that the two cell types are sensing extracellular Ca2+ through the same Ca2+-sensing receptor, a fundamental different mechanism for processing of the Ca2+ signal in both cells must be postulated. Whereas in parathyroid cells extracellular Ca2+-induced elevations of cytosolic Ca2+ are due to Ca2+ release from intracellular stores and subsequently to influx by voltage-independent Ca2+ channels, in C-cells the release from internal stores appears to play only a small role. Cytosolic Ca2+ elevations in the C-cells appear mainly to be due to Ca2+ influx from voltage-dependent Ca2+ channels (Scherubl et al. 1991 ). It is interesting that C-cells of the human thyroid gland are devoid of PV, whereas the distribution of PV in the kidney (Schneeberger and Heizmann 1986 ) matches fairly well with the distribution of CaR (Riccardi et al. 1995 ) in the distal tubule and proximal collecting duct, where the fine regulation of Ca2+ readsorption takes place. CaR is also present in the cortical thick ascending limb, the proximal tubule, and the medullar ascending limb, where PV has not been found.

Finally, the presence of CaR is not limited only to cells involved in mineral ion metabolism but has also been found in brain (Ruat et al. 1995 ). A comparison of PV expression and CaR expression in brain has yet to be established.

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.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Bading H, Ginty DD, Greenberg ME (1993) Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260:181-186[Medline]

Berchtold MW (1989) Structure and expression of genes encoding the three-domain Ca2+-binding proteins parvalbumin and oncomodulin. Biochim Biophys Acta 1009:201-215[Medline]

Brown EM (1991) Extracellular Ca2+ sensing regulation of parathyroid cell function, and role of Ca2+ and other ions as extracellular (first) messengers. Physiol Rev 71:371-411[Free Full Text]

Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC (1993) Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 366:575-580[Medline]

Celio MR, Baier W, Scharer L, de Viragh PA, Gerday C (1988) Monoclonal antibodies directed against the calcium binding protein parvalbumin. Cell Calcium 9:81-86[Medline]

Chard PS, Bleakman D, Christakos S, Fullmer CS, Miller RJ (1993) Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones. J Physiol (Lond) 472:341-357[Abstract]

Davidoff MS, Schulze W, Middendorff R, Holstein AF (1993) The Leydig cell of the human testis—a new member of the diffuse neuroendocrine system. Cell Tissue Res 271:429-439[Medline]

de Lellis RA (1993) Tumors of the parathyroid gland. In Atlas of Tumor Pathology Series. Washington, Armed Forces Institute of Pathology, 1-14

Doglioni C, Dell'Orto P, Coggi G, Iuzzolino P, Bontempini L, Viale G (1987) Choroid plexus tumors. An immunocytochemical study with particular reference to the coexpression of intermediate filament proteins. Am J Pathol 127:519-529[Abstract]

Dreessen J, Lutum C, Schafer BW, Heizmann CW, Knopfel T (1996) Alpha-parvalbumin reduces depolarization-induced elevations of cytosolic free calcium in human neuroblastoma cells. Cell Calcium 19:527-533[Medline]

Fried RM, Tashjian AHJ (1986) Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells. J Biol Chem 261:7669-7674[Abstract/Free Full Text]

Föhr U, Weber BR, Müntener M, Fohr UG, Weber BR, Muntener M, Staudenmann W, Hughes GJ, Frutiger S, Banville D, Schafer BW, Heizmann CW (1993) Human {alpha} and ß parvalbumins: structure and tissue-specific expression. Eur J Biochem 215:719-727[Abstract]

Garrett JE, Capuano IV, Hammerland LG, Hung BC, Brown EM, Hebert SC, Nemeth EF, Fuller F (1995) Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem 270:12919-12925[Abstract/Free Full Text]

Heizmann CW, Berchtold MW (1987) Expression of parvalbumin and other Ca2+-binding proteins in normal and tumor cells: a topical review. Cell Calcium 8:1-41[Medline]

Hurwitz S (1996) Homeostatic control of plasma calcium concentration. Crit Rev Biochem Mol Biol 31:41-100[Abstract]

Itzhaki RF, Gill DM (1964) A micro-biuret method for estimating proteins. Anal Biochem 9:401-410

Kägi U, Berchtold MW, Heizmann CW (1987) Ca2+-binding parvalbumin in rat testis. J Biol Chem 262:7314-7320[Abstract/Free Full Text]

Kawasaki H, Kretsinger RH (1994) Calcium-binding proteins 1: EF-hands. Protein Profile 1:343-517[Medline]

Kretsinger RH (1980) Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem 8:119-174[Medline]

Lundgren S, Hjalm G, Hellman P, Ek B, Juhlin C, Rastad J, Klareskog L, Akerstrom G, Rask L (1994) A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily. Exp Cell Res 212:344-350[Medline]

Maruyama K, Mikawa T, Ebashi S (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem (Tokyo) 95:511-519[Abstract]

Miki H, Maercklein PB, Fitzpatrick LA (1995) Spontaneous oscillations of intracellular calcium in single bovine parathyroid cells may be associated with the inhibition of parathyroid hormone secretion. Endocrinology 136:2954-2959[Abstract]

Moncrief ND, Kretsinger RH, Goodman M (1990) Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J Mol Evol 30:522-562[Medline]

Müller BK, Kabos P, Belhage B, Neumann T, Kater SB (1996) Transfected parvalbumin alters calcium homeostasis in teratocarcinoma PCC7 cells. Eur J Cell Biol 69:360-367[Medline]

Nakayama S, Moncrief ND, Kretsinger RH (1992) Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories. J Mol Evol 34:416-448[Medline]

Nitsch C, Scotti A, Sommacal A, Kalt G (1989) GABAergic hippocampal neurons resistant to ischemia-induced neuronal death contain the Ca2(+)-binding protein parvalbumin. Neurosci Lett 105:263-268[Medline]

Pauls TL, Cox JA, Berchtold MW (1996) The Ca2+(-)binding proteins parvalbumin and oncomodulin and their genes: new structural and functional findings. Biochim Biophys Acta 1306:39-54[Medline]

Peunova N, Enikolopov G (1993) Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells. Nature 364:450-453[Medline]

Pietrobon D, Di Virgilio F, Pozzan T (1990) Structural and functional aspects of calcium homeostasis in eukaryotic cells. Eur J Biochem 193:599-622[Abstract]

Riccardi D, Park J, Lee WS, Gamba G, Brown EM, Hebert SC (1995) Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci USA 92:131-135[Abstract]

Ridefelt P, Bjorklund E, Akerstrom G, Olsson M J, Rastad J, Gylfe E (1995) Ca(2+)-induced Ca2+ oscillations in parathyroid cells. Biochem Biophys Res Commun 215:903-909[Medline]

Ruat M, Molliver ME, Snowman AM, Snyder SH (1995) Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals. Proc Natl Acad Sci USA 92:3161-3165[Abstract]

Scherubl H, Schultz G, Hescheler J (1991) Electrophysiological properties of rat calcitonin-secreting cells. Mol Cell Endocrinol 82:293-301[Medline]

Schneeberger PR, Heizmann CW (1986) Parvalbumin in rat kidney. Purification and localization. FEBS Lett 201:51-56[Medline]

Schwaller B, Dick J, Dhoot G, Carroll S, Vrbova G, Nicotera P, Pette D, Wyss A, Bluethmann H, Hunziker W, Celio MR (1999) Prolonged contraction-relaxation cycle of fast-twitch muscles in parvalbumin knockout mice. Am J Physiol 276:C395-403[Abstract/Free Full Text]

Wnuk W, Cox JA, Stein EA (1982) Parvalbumins and other soluble high-affinity calcium binding proteins from muscle. In Cheung WY, ed. Calcium and Cell Function. Vol 2. New York, Academic Press, 243-278

Yoshida A, Ueda T, Takauji R, Liu YP, Fukushima T, Inuzuka M, Nakamura T (1993) Role of calcium ion in induction of apoptosis by etoposide in human leukemia HL-60 cells. Biochem Biophys Res Commun 196:927-934[Medline]





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Pauls, T. L.
Articles by Celio, M. R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Pauls, T. L.
Articles by Celio, M. R.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]