ARTICLE |
Correspondence to: René J.M. Bindels, 160 Cell Physiology, University Medical Centre Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands. E-mail: reneb@sci.kun.nl
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Summary |
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The epithelial Ca2+ channel, ECaC1, is primarily expressed in the apical membrane of vitamin D-responsive tissues. This study characterizes for the first time the presence of this novel channel in pancreatic tissue by reverse transcriptase-polymerase chain reaction and immunohistochemistry. In addition, the expression of ECaC1 was investigated in an animal model for Type 2 diabetes mellitus, the Zucker diabetic fatty (ZDF) rat. Identical staining patterns for ECaC1 and insulin were observed, whereas no co-localization of ECaC1 with glucagon was found. ECaC1, insulin, and prohormone convertase 1 (a neuroendocrine endoprotease expressed in secretory granules) showed a similar punctate staining. ECaC1 co-localized with the Ca2+ binding protein calbindin-D28K in the ß-cells. Furthermore, in contrast to wild-type rats, in ZDF rats aging led to a progressive decrease in both insulin and ECaC1 staining. Plasma 1,25-dihydroxyvitamin D3 levels were similar in both control and ZDF rats and decreased with aging. Taken together, our findings indicate that this novel Ca2+ channel may play a role in the regulation of endocrine Ca2+ homeostasis. (J Histochem Cytochem 50:789798, 2002)
Key Words: ß-cell, calbindin-D28K, 1,25-dihydroxyvitamin D3, NIDDM, ZDF rat
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Introduction |
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THE epithelial Ca2+ channel ECaC1 was recently cloned from rabbit kidney and was thought to play a crucial role in Ca2+ homeostasis as gatekeeper of active transcellular Ca2+ transport (
In pancreatic ß-cells, calcium plays a central role in stimulussecretion coupling and is regulated by glucose and other secretagogues. An increase in intracellular Ca2+ concentrations ([Ca2+]i) is necessary to induce insulin secretion (
As in kidney, in pancreatic ß-cells Na+/Ca2+ exchanger (NCX), parathyroid hormone-related peptide (PTHrP), calbindin-D28K, and receptors for 1,25-dihydroxyvitamin D3 have been found (
The aim of the present study was to investigate by RT-PCR and immunohistochemistry whether ECaC1 is expressed in rat pancreatic tissue. In our studies, we included the Zucker diabetic fatty (ZDF) rat, an animal model for Type 2 DM. The ZDF rat becomes mildly hyperglycemic between 7 and 10 weeks of age and thereafter spontaneously develops overt diabetes (
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Materials and Methods |
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Experimental Animals
Male Zucker diabetic fatty (ZDF/Gmi-fa/fa or ZDF) and lean Zucker control (ZDF/Gmi-+/? or control) rats were obtained from Genetic Models (Indianapolis, IN). All rats were maintained on a 12-hr light/dark cycle (lights on at 0700, lights off at 1900 hr), and had ad libitum access to tapwater and chow (RMH-B: 22.8% protein, 5.1% fat, 4.2% fiber and 67.9% other carbohydrates; Hope Farms, Woerden, The Netherlands). Animals were studied at 6, 12, and 24 weeks of age. For immunofluorescence studies, three control and three ZDF rats were examined at each time point. All animal experiments were performed according to the principles of laboratory animal care (NIH publication no. 85-23, revised 1985) and Dutch laws on the protection of animals.
Blood Sampling and 1,25-Dihydroxyvitamin D3 Determination
Blood samples for 1,25-dihydroxyvitamin D3 determinations were obtained from control and ZDF rats under isoflurane anesthesia at 6, 12, 21, and 24 weeks of age by orbital puncture and collected in precooled tubes containing dry lithiumheparin additive (30 UP U/tube; Vacutainer, Becton Dickinson, Etten-Leur, The Netherlands). Tubes were gently shaken and centrifuged for 10 min at 1500 x g (4C). Plasma samples were stored at -80C until assayed. Plasma 1,25-dihydroxyvitamin D3 levels were measured using a radioreceptor assay after extraction of the samples, followed by paper chromatography as described previously (
RT-PCR
Under isoflurane anesthesia of the rats, the pancreas was rapidly removed and cleared of fat. Small parts of the pancreas were immediately frozen in liquid nitrogen. For RNA isolation, the tissue was ground with a mortar and pestle that were pretreated with 70% ethanol in diethyl pyrocarbonate (DEPC; Sigma Chemical, St Louis, MO). Total RNA was isolated from the resulting powder using TRIzol (Gibco BRL; Gaithersburg, MD) according to the manufacturer's instructions, except that an additional acidic phenolchloroform extraction was performed to remove excessive protein. For cDNA synthesis, 25 µg of total RNA was incubated with 5 mU Pd(N)6 (Pharmacia; Roosendaal, The Netherlands) at 70C for 10 min. Thereafter, the RNA was reverse transcribed (RT) in a final volume of 20 µl containing 10 mM dithiothreitol, 0.5 mM dNTPs, 40 U RNase inhibitor (Promega; Madison, WI), and 100 U of Superscript II (Gibco BRL) for 1.5 hr at 37C. The samples were then incubated at 70C for 10 min and subsequently diluted to a final volume of 50 µl. For RT-PCR analysis, 2.5 µl of cDNA was used as a template in a 25-µl amplification mixture containing Sybr Green (PE Applied Biosystems Benelux; Nieuwerkerk a/d IJssel, The Netherlands), 1 mM dNTPs, 3 mM MgCl2, 0.6 U Taq Gold polymerase (PE Applied Biosystems) and 0.6 µM of each primer. The primer sets used in the PCR were: for insulin (118-bp fragment), sense 5'-gggaacgtggtttcttctacaca-3', antisense 5'-tccagtgccaaggtctgaagat-3'; for ECaC1 (158 bp), sense 5'-agcaatagccaccgtggatg-3', antisense 5'-atgtcccagggtgtttcgac-3'; for calbindin-D28K (70 bp), sense 5'-ggagctgcagaacttgatccag-3', antisense 5'-ctcaggtgatagctccaatccag-3'; and for ß-actin (84 bp), sense 5'-accctaaggccaaccgtga-3', antisense 5'-cagcctggatggctacgtacat-3'. After an initial step at 95C for 10 min, a real-time semiquantitative PCR of 40 cycles was performed (PE Applied Biosystems), each cycle consisting of 15-sec denaturation at 95C and 1-min annealing at 60C. Cycle threshold (CT) values were determined using the PE Applied Biosystems software. CT is defined as the cycle number at which the amount of amplified target passes a fixed threshold above baseline. As a control for the amount of RNA used, ß-actin was used.
Antisera
The guinea pig anti-rabbit ECaC1 polyclonal antiserum was directed against a peptide corresponding to the 10 carboxy-terminal amino acid residues 698707 (NH2-SHRGWEILRQ -COOH) of ECaC1. Anti-insulin and anti-glucagon polyclonal antisera were obtained from Dakopatts (Copenhagen, Denmark). The anti-prohormone convertase 1 (PC1) polyclonal antiserum was kindly provided by Dr. W.J.M. Van de Ven (Leuven, Belgium) and has been extensively characterized (
Immunofluorescence Studies
Under isoflurane anesthesia of the rats, the pancreas was rapidly removed and cleared of fat. Small parts of the pancreas were fixed overnight at 4C in 4% phosphate-buffered formaldehyde (pH 7.4). The tissues were then rinsed in PBS, dehydrated in a graded series of ethanol, rinsed in xylene two times for 30 min each, and impregnated twice for 30 min with Paraplast Plus (Oxford Labware; St Louis, MO) under vacuum. Paraffin sections 4 µm thick were mounted on SuperFrost Plus glass slides (Merck; Darmstadt, Germany) and kept overnight at 37C. For immunofluorescence studies, tissue sections were dewaxed with two changes of xylene, rehydrated through graded ethanol, and then washed with PBS (pH 7.4). Thereafter, sections were boiled for 2 min in citrate buffer (0.01 M C5H6Na3O7 and 0.01 M C6H8O7, pH 6), cooled to room temperature (RT), and thoroughly rinsed with TN buffer (0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5). Sections were incubated with TNB buffer for 30 min before overnight incubation at 4C with the primary antibodies. Then the sections were rinsed three times for 5 min in TNT buffer [TN-buffer containing 0.05% (v/v) Tween-20]. Sections stained for ECaC1 and insulin were incubated for 1 hr at RT with goat anti-guinea pig Alexa 594-conjugated anti-IgG (1:300) (Molecular Probes; Eugene, OR). Sections double stained for ECaC1 and glucagon were incubated with both goat anti-guinea pig Alexa 594-conjugated anti-IgG (1:300) and goat anti-rabbit Alexa 488-conjugated anti-IgG (1:300) (both from Molecular Probes). The sections double stained with ECaC1 and PC1 or with ECaC1 and calbindin-D28K were incubated for 30 min at RT with pig anti-rabbit biotinylated antiserum (1:1500) (NEN Life Science Products; Boston, MA), rinsed three times in TNT buffer, and then incubated for 30 min at RT with streptavidinHRP antiserum (1:100) (NEN Life Science Products). After three 5-min rinses in TNT buffer, the peroxidase was visualized by incubation with fluorescent tyramides (1:50 in amplification solution) (NEN Life Science Products) for 7 min. For detection of ECaC1, these double stained sections were incubated for 1 hr at RT with goat anti-guinea pig Alexa 594-conjugated anti-IgG (1:300) (Molecular Probes). All sections were rinsed thoroughly with TNT buffer, rinsed in graded methanol, and mounted in Mowiol (Hoechst; Frankfurt, Germany) containing 2.5% (w/v) NaN3. Photographs were taken with a Zeiss Axioskop microscope equipped for epifluorescence illumination using Kodak EPH P1600X film.
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Results |
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ECaC1 mRNA Expression in Rat Pancreatic Tissue
To establish if ECaC1 is expressed in the rat pancreas, we performed RT-PCR analysis on RNA isolated from total pancreatic tissue of 6-week-old control and ZDF rats. In addition, mRNA expression of insulin and calbindin-D28K was analyzed in the pancreas of both control and ZDF rats. In control rats, mRNA expression was found for ECaC1 (CT 33 ± 1), insulin (CT 21 ± 1), calbindin-D28K (CT 34 ± 1), and ß-actin (CT 22 ± 1). In addition, between control and ZDF rats no significant differences in CT values were observed, suggesting that mRNA expression levels of these proteins were similar in both rat strains. No amplification was found when the RT step was omitted or in the water controls. Analysis of ß-actin mRNA was used as a control for RNA isolation and cDNA synthesis.
Immunofluorescence Localization of ECaC1 in Rat Pancreatic Islets
To localize ECaC1 protein expression in pancreatic tissue, immunofluorescence experiments were performed. In the rat pancreas, identical labelling patterns were found for ECaC1 and insulin, i.e., immunopositive staining was present in the pancreatic islets, whereas no staining was observed in the exocrine pancreas (Fig 1A). At higher magnification, punctate ECaC1 and insulin staining was found within the cytoplasm of the endocrine cells, suggesting a localization of ECaC1 in the secretory granules of the ß-cells (Fig 1B). Sections incubated with ECaC1 antiserum pre-absorbed for 1 hr with ECaC1 peptide or pre-immune antiserum were devoid of any immunostaining (Fig 2D, left and middle columns).
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To analyze in more detail the localization of ECaC1 in the pancreatic islets, we performed double immunofluorescence studies. Labeling of rat pancreatic tissue with anti-ECaC1 and anti-glucagon antibodies revealed that ECaC1 did not co-localize with glucagon, indicating that ECaC1 is not expressed in pancreatic -cells (Fig 2A). Double immunofluorescence experiments showed complete co-localization in the ß-cells of ECaC1 and PC1, a neuroendocrine endoprotease expressed in secretory granules (Fig 2B). In agreement with previous studies (
- and ß-cells, with the clearest staining being found in the
-cells. Double staining of ECaC1 with calbindin-D28K revealed their co-localization in the ß-cells but not in the
-cells, suggesting that, in the pancreas, ECaC1 expression is restricted to the ß-cells (Fig 2C). A control was performed in which as secondary antibody a goat anti-rabbit IgG was employed in combination with the guinea pig anti-ECaC1 antibody as the primary antibody to detect possible crossreactivity (Fig 2D, right). No immunopositive staining was observed under these experimental conditions and crossreactivity can therefore be excluded.
ECaC1 Expression During the Progression of Diabetes in ZDF Rats
Because glucose-induced insulin secretion is impaired in Type 2 DM, probably due to the inability of glucose to increase intracellular Ca2+ concentrations, we studied the expression of ECaC1 in ZDF rats, an animal model for Type 2 DM. In control rats between 6 and 24 weeks of age, no difference in the amount of immunostaining of both ECaC1 and insulin was observed (Fig 3A). In 6-week-old ZDF rats, the amount of ECaC1 and insulin staining was about the same as that in control rats of the same age (Fig 3B). However, in ZDF rats, aging affected the staining patterns of both ECaC1 and insulin, i.e., from 12 weeks onwards a decrease in the amount of immunostaining of these two proteins was observed. At 24 weeks of age the amount of insulin staining had progressively decreased in the ZDF rat compared to that in 6-week-old ZDF rats. Remarkably, the amount of immunostaining of ECaC1 in 24-week-old ZDF rats was also decreased compared to the 6-week-old ZDF rats (Fig 3B).
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Plasma 1,25-Dihydroxyvitamin D3 Concentrations in Control and ZDF Rats
To establish if the reduction of ECaC1 expression in the ZDF rats was accompanied by a change in 1,25-dihydroxyvitamin D3 levels, we measured plasma vitamin D concentrations in both control and ZDF rats from 6 to 24 weeks of age. Plasma 1,25-dihydroxyvitamin D3 levels were identical in control and ZDF rats and decreased with aging (Fig 4). At 6 weeks of age, in control and ZDF rats 1,25-dihydroxyvitamin D3 levels were 548 ± 15 pmol/liter and 556 ± 27 pmol/liter, respectively. These levels decreased progressively to 295 ± 26 pmol/liter and 320 ± 20 pmol/liter in control and ZDF rats of 24 weeks of age, respectively, showing that although a decrease in plasma vitamin D concentrations was observed in the ZDF rats, the decrease in ECaC1 expression in these rats was not due to a reduction in vitamin D levels (Fig 4).
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Discussion |
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In pancreatic ß-cells, calcium plays an important role in insulin secretion since an increase in [Ca2+]i is a prerequisite to facilitate stimulussecretion coupling. At present, the complex calcium-mediated transduction pathway in the ß-cells is not clear, but a close interplay between plasma membrane ion channels and secretory granules may be needed (
The secretory granules of ß-cells contain high levels of Ca2+ implicated in the sorting and processing of precursor proteins (
In pancreatic ß-cells, the maintenance of intracellular Ca2+ homeostasis is a complex process because in these cells many proteins, such as NCX, PTHrP, calbindin-D28K, receptors for 1,25-dihydroxyvitamin D3, and now also ECaC1, have been found that are postulated to play a role in Ca2+ homeostasis and insulin release. On the basis of the analysis of calbindin-D28K knockout mice, -cells vitamin D negatively regulates both calbindin-D28K expression and glucagon release, suggesting that the role of calbindin and vitamin D in Ca2+ handling is different in
- and ß-cells. In agreement with the study of
- and ß-cells, the clearest staining being present in the
-cells. Thus far, ECaC1 has always been found to co-localize with calbindin-D (
-cells suggests that the modulation of [Ca2+]i, in which both ECaC1 and calbindin-D28K may play a role, is different in
- and ß-cells. This is in line with the notion of
In ZDF rats, progression of diabetes is accompanied by a reduced expression of insulin (
As already mentioned, vitamin D together with calcium may modulate ß-cell function. Our data show that aging of both control and ZDF rats is accompanied by a decrease in plasma 1,25-dihydroxyvitamin D3 levels, which confirms previous observations (
In conclusion, ECaC1 is abundantly expressed in rat pancreatic islets. Punctate cytoplasmic ECaC1 expression was restricted to the ß-cells, suggesting a localization of ECaC1 to the membranes of secretory granules. We propose a function of ECaC1 in the maintenance of intracellular Ca2+ homeostasis either as part of a Ca2+ efflux pathway from granules to the cytoplasm or as a component of a Ca2+ influx pathway for extracellular Ca2+ into the cytoplasm. Furthermore, in ZDF rats a diabetes-related reduction of ECaC1 expression, independent of plasma 1,25-dihydroxyvitamin D3 levels, was demonstrated. This decrease in ECaC1 expression in the ZDF rat may contribute to the impaired stimulussecretion coupling observed in diabetes. Altogether, the present study suggests that ECaC1 may play an important role in the regulation of Ca2+ homeostasis in pancreatic ß-cells.
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Acknowledgments |
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Supported by grant #95.116 from the Dutch Diabetes Foundation (Diabetes Fonds Nederland). JGJ Hoenderop was supported by a long-term EMBO fellowship (ALTF 160-2000). This work was also supported by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001).
We are grateful to Dr S. Christakos (New Jersey) and Dr W.J.M van de Ven (Leuven, Belgium) for kindly providing antisera against calbindin-D28K and PC1, respectively. We also thank Annet Verleg and Henk Arnts (Central Animal Laboratory; University of Nijmegen, The Netherlands) for biotechnical assistance, Rob van den Berg (Department of Chemical Endocrinology; University Medical Centre Nijmegen, The Netherlands) for plasma 1,25-dihydroxyvitamin D3 measurements, and Anita Hartog (Department of Cell Physiology; University of Nijmegen, The Netherlands) for assistance in performing the immunofluorescence techniques.
Received for publication October 29, 2001; accepted December 27, 2001.
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