ARTICLE |
Correspondence to: Toni Schneider, Univ. of Cologne, Institute of Neurophysiology, Robert-Koch-Str. 39, D-50931 Köln, Germany. Fax 49-221-4786965.
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Summary |
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Polyclonal antibodies were raised against a common and a specific epitope present only in longer 1E isoforms of voltage-gated Ca2+ channels, yielding an "anti-E-com" and an "anti-E-spec" serum, respectively. The specificity of both sera was established by immunocytochemistry and immunoblotting using stably transfected HEK-293 cells or membrane proteins derived from them. Cells from the insulinoma cell line INS-1, tissue sections from cerebellum, and representative regions of gastrointestinal tract were stained immunocytochemically. INS-1 cells expressed an
1E splice variant with a longer carboxy terminus, the so-called
1Ee isoform. Similarily, in rat cerebellum, which was used as a reference system, the anti-E-spec serum stained somata and dendrites of Purkinje cells. Only faint staining was seen throughout the cerebellar granule cell layer. After prolonged incubation times, neurons of the molecular layer were stained by anti-E-com, suggesting that a shorter
1E isoform is expressed at a lower protein density. In human gastrointestinal tract, endocrine cells of the antral mucosa (stomach), small and large intestine, and islets of Langerhans were stained by the anti-E-spec serum. In addition, staining by the anti-E-spec serum was observed in Paneth cells and in the smooth muscle cell layer of the lamina muscularis mucosae. We conclude that the longer
1Ee isoform is expressed in neuroendocrine cells of the digestive system and that, in pancreas,
1Ee expression is restricted to the neuroendocrine part, the islets of Langerhans.
1E therefore appears to be a common voltage-gated Ca2+ channel linked to neuroendocrine and related systems of the body. (J Histochem Cytochem 47:981993, 1999)
Key Words:
1E calcium channels, cerebellum, INS-1 insulinoma cell line, secretion, endocrine digestive system, islets of Langerhans, chromogranin A, gastric antrum, small intestine, large intestine, Paneth cells, immunocytochemistry
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Introduction |
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VOLTAGE-GATED Ca2+ CHANNELS are potent signal transducers during muscle contraction, secretion, and a variety of neuronal processes including excitability, neurotransmitter release, synaptic plasticity, and gene expression (1-subunit and several auxiliary subunits, including the
2
- and ß-subunits (
-subunits are known for skeletal muscle (
Ten different 1-subunits of voltage-gated Ca2+ channels have been cloned thus far (Table 1) and are classified according to their voltage dependence of activation as high voltage- (
1S,
1C,
1D,
1F, and
1A,
1B,
1E) or low voltage-activated Ca2+ channel types (
1G,
1H, and
1I) (
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High voltage-activated Ca2+ channels can be further subdivided as a dihydropyridine-sensitive L-type subfamily containing 1S,
1C, and
1D Ca2+ channels (Table 1). The recently identified retinal
1F subunit is structurally related to the L-type subfamily but not yet functionally characterized in detail (
The non-L-type subfamily of high voltage-activated Ca2+ channels consists of 1A-,
1B-, and
1E-subunits which are pharmacologically divergent. Two of them,
1A and
1B, have similar physiological functions. They are both involved in hormonal modulation of presynaptic Ca2+ currents (
1A and
1B (
1-subunits,
1A,
1B, and
1E, have been intensively investigated for their G-protein-dependent modulation (as summarized in
The physiological function of 1E-containing voltage-gated Ca2+ channels is not yet well understood. The
1E-subunit has been related to the resistant (R)-type Ca2+ channels from cerebellar granule cells (
1E antisense oligonucleotides (
1E isoform (
1E has been analyzed in detail by using antibodies raised against common regions of individual
1E-subunits (
Neurons and endocrine cells that are responsible for the production of polypeptide hormones are developmentally and functionally related to each other (
A new isoform of 1E was isolated from rat pancreas and human kidney by amplifying cDNA fragments of
1E (
1Ee" for this splice variant according to the guidelines of nomenclature for voltage-gated Ca2+ channels (
1E isoforms. The cDNA fragments of longer isoforms of
1E have been detected recently in mouse, rat, and human cerebellum (
Our recent RT-PCR results from isolated islets of Langerhans (1E isoform is expressed in several peripheral organs. This led to the hypothesis that other endocrine systems might contain
1E. Therefore, we investigated the expression of the neuronal
1E calcium channel isoforms in the central nervous system and in selected areas of the human gut: the antrum of the stomach, the small and large intestine, and the islets of Langerhans isolated from pancreas. In addition, the rat insulinoma cell line INS-1 was included both in the immunohistochemical investigation and electrophysiological studies to establish the physiological function of
1E in this cell line and in related tissues.
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Materials and Methods |
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Materials
The ECL detection kit for immunoblotting was purchased from Amersham (Braunschweig, Germany) and collagenase P from Boehringer Mannheim (Germany). The monoclonal antibodies (MAbs) against human chromogranin A and human insulin were purchased from Camon (Wiesbaden, Germany) and BioGenex (DCS; Hamburg, Germany), respectively. The polyclonal antibody against human somatostatin was a kind gift from BioGenex. All reagents were of standard biochemical quality.
Cell Lines and Tissues
All procedures used in the present study that involved tissues from animals and humans were performed in accordance with the regulations governing their use in scientific research.
Aliquots of the INS-1 cell line were a gift from Dr. S. Ullrich with the kind permission of Dr. C.B. Wollheim (Geneva). The INS-1 cells are grown in RPMI 1640 medium, 10% heat-inactivated fetal calf serum, 50 µM 2-mercaptoethanol, 1 mM Na-pyruvate, 10 mM Hepes, pH 7.27.5, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Stable HEK-293 cell lines expressing human Ca2+ channel 1E-subunits (
1Ed cDNA or a deletion mutant of
1Ed (ß1Ed-CDEL) lacking a 129-bp insert of the 3'-end (
Antibody Production
Two different peptides were used for the immunization of rabbits. Peptide Nast-195 is homologous to a sequence close to the pore region in Domain I and therefore corresponds to a common sequence in all published 1E isoforms. Peptide Nast-197 is part of the 43-amino-acid (aa) extension cloned within the fetal brain
1Ed isoform (
1E isoforms,
1Ee and
1Ef (
1Ed isoform) were synthesized by the solid-phase method (
The sequences of the synthetic peptides were compared with the aa sequences in GenBank by using the program blastp 2.0.4 (1E fragments cloned from mammalian species were picked up and shown to be similar or identical to the synthetic peptide sequences. The peptide sequence of Nast-195 is identical to the rat, mouse, and all published human sequences, whereas the rabbit
1E shows a single amino acid substitution of valine for isoleucine. For the peptide Nast-197, both the rat and the mouse sequence show one aa difference with an additional proline instead of a serine residue compared to the human
1E sequence. However, no differences were observed during immunostaining comparing human and rat cerebellum or pancreas. Both sera do not stain the
1G- or the
1H-subunit in membranes from stably transfected HEK-293 cell lines (unpublished results). Therefore, the anti-E-com serum (anti-Nast-195) is used to detect all known
1E isoforms and the anti-E-spec serum (anti-Nast-197) detects the longer
1Ed,
1Ee, and
1Ef or structurally related isoforms containing a longer carboxy terminus (
1E isoforms in the cerebellum, both sera were purified by adsorption to liver powder to reduce any unspecific staining. Rat liver (6 g) was homogenized in cold PBS, pH 7.4 (6 ml), and proteins were precipitated by adding a fourfold volume of acetone. After 30 min on ice, the unsoluble proteins were separated by centrifugation for 10 min at 10,000 x g. The precipitated proteins were washed once in 5 ml ice-cold acetone by rehomogenization and precipitated by centrifugation as before. The protein pellet was spread on filter paper and, after drying, homogenized to a powder, which was stored in aliquots at -20C. The peptide-specific antisera (0.5 ml of 1:50 diluted crude sera) were incubated with 20 mg of liver powder for 1 hr at 37C and centrifuged for 10 min at 2000 x g. The supernatant was sterile-filtered through 0.2-µm filters and used for immunohistochemistry of rat cerebellar sections.
Preparation of Paraffin-processed Tissue
Female Wistar rats weighing 180250 g were anesthetized with CO2, decapitated, and the organs rapidly removed. The individual tissues were dissected into smaller blocks and fixed by immersion in 10% neutral buffered formalin at room temperature (RT) overnight. Tissue sections of human gastric antrum, small and large intestine (cecum), and pancreas were obtained from surgically resected specimens. All tissues, including rat cerebellum, were fixed in 10% buffered formalin and embedded in paraffin. Immunohistochemistry was performed on 4-µm deparaffinized serial tissue sections that were mounted on silane-coated slides and dried at 50C before staining.
Western Blot Analysis and Immunocytochemical Staining of Cell Lines and Tissues
The sensitivity and specificity of the anti-1E sera were tested on immunoblots of microsomal membrane proteins from stably transfected HEK-293 cells. These cells were transfected either with the full-length human
1Ed (
1Ed-CDEL lacking a 129-bp insertion of the carboxy terminus, which carries the region of the epitope for anti-E-spec (
In addition, the specificity of the sera was tested by immunostaining untransfected and stably transfected HEK-293 cells expressing human 1E (
1E, the cells were treated with a standard streptavidinbiotinhorseradish peroxidase technique according to the instructions of the manufacturer (BioGenex). The primary antibody was diluted 1:500 or 1:1000 in PBS containing 2% BSA.
For immunohistochemistry, the polyclonal antibodies anti-E-com and anti-E-spec were used at a working dilution of 1:20 (in several tissues as indicated), 1:50 (cerebellum), or 1:200 (large intestine). Before immunostaining, sections underwent a microwave-based heat-induced epitope retrieval (HIER) treatment in a solution of 10 mM citrate buffer, pH 6.0. Endogenous peroxidase activity was quenched by exposure to a solution of 0.3% hydrogen peroxide in methanol for 20 min at 37C. After washing with PBS, the sections were immunostained by the streptavidinbiotinhorseradish peroxidase technique according to the instructions of the manufacturer (Super Sensitive detection system from BioGenex). 3,3'-Diaminobenzidine (DAB) was used as chromogen with and without copper sulfate enhancement. Two negative control procedures were performed. Either the primary antibodies were replaced by the preimmune serum or the serum was preincubated with an excess of the peptide (20 µM) used for immunization. Sections from a multitissue block were used to verify appropriate positive and negative reactions.
In some experiments the purified anti-E serum directed against an 1E fusion protein was used as a positive control (
Electrophysiological Recordings
Electrophysiological recordings from INS-1 cells were done in the whole-cell configuration of the patch-clamp technique at RT (2022C) with an Axopatch 200 patch-clamp amplifier (Axon Instruments; Burlingame, CA). The bath solution contained (in mM) 15 BaCl2, 130 N-methyl-glucamine, 10 HEPES, 5 KCl, pH adjusted with HCl to 7.4. The patch electrodes had resistances of 2-4 M when filled with an electrode solution composed of (in mM) 130 CsCl, 5 oxalacetate, 5 creatine, 5 pyruvate, 1 EGTA, 10 HEPES/CsOH (pH 7.4). The cells were placed on a 12-mm-wide round coverslip in a recording chamber and were continuously superfused at a rate of 25 ml/min driven by gravity from several reservoirs. The membrane currents were corrected for leakage and capacitive currents on line using a P/4 subpulse protocol (ISO2; MFK, Germany). Capacitance of a cell was estimated by means of ramp depolarizations. Currents were filtered at 1 kHz and digitized at 5 kHz. Currents were measured either immediately after breakthrough or at 1 or 2 min later after establishment of the whole-cell configuration of the patch-clamp technique.
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Results |
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T-type and Non-L-type Currents in the Insulinoma Cell Line INS-1
The INS-1 cell line is a favored model system for insulin secretion (1E-subunit was detected in insulinoma cell lines and islets of Langerhans by RT-PCR using specific oligonucleotide primer pairs (
1E cDNA fragments were amplified (
1Ed Ca2+ channel subunit in HEK-293 cells (unpublished results). Low and high voltage-activated Ca2+ currents, not related to L-type currents, are clearly detected in INS-1 cells, as shown by a representative example (Figure 1A). With Ba2+ as charge carrier, fast-activating transient inward currents were observed, which are later superimposed by high voltage-activated Ca2+ currents that arise during run-up (Figure 1B). INS-1 cells clearly express dihydropyridine-insensitive voltage-gated Ca2+ currents which might contain resistant components related to
1E.
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Specificity and Sensitivity of the Sera
Two 1E-specific sera were used for immunostaining of
1E Ca2+ channels. The anti-E-com serum has been designed to recognize all cloned mammalian
1E-isoforms, while the anti-E-spec serum is directed against part of the 43 amino acid long insert III (
1E isoforms,
1Ed,
1Ee, and
1Ef (
1E expressing HEK-293 cells (
1Ed-CDEL were analyzed lacking the peptide epitope (peptide Nast-197) used for raising antibodies (
The untransfected HEK-293 cells were negative for the peptide antigens, whereas the 1Ed-transfected HEK-293 cells reacted strongly positive with the anti-E-spec serum (Figure 2A). In the insulinoma cell line INS-1, the positive staining using anti-E-spec was blocked by preincubation of the serum with the peptide Nast-197, which was used originally for immunization of the rabbits (Figure 2B). However, positive staining could be observed with the anti-E-spec serum (Figure 2C), which corresponds to recent RT-PCR results (
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Localization of Longer 1E Isoforms in Rat Cerebellum
In human cerebellar Purkinje cells, expression of 1E has been detected by use of common anti-
1E antibodies (
1E transcripts detected by in situ hybridization was reported to be below the limits of detection in rat cerebellar Purkinje cells (
1E isoforms in rat cerebellum was investigated in the present study.
Longer 1E isoforms were detected in the somata and axons of rat Purkinje cells using the anti-E-spec serum (Figure 2D). Similar results were obtained with the common antibody (Figure 2E). When the incubation time with the anti-E-com serum was prolonged, additional positive neurons were detected in the molecular layer of rat cerebellum (Figure 2F). There was no staining when preimmune serum was used instead of primary antibody (not shown) or when the serum was preincubated with the corresponding peptide (Figure 2G). Hence, the immunolocalization of
1E shows that longer
1E isoforms are expressed in rat cerebellum in a pattern similiar to that reported for human cerebellum (
1E isoforms at lower protein density. The identification of a shorter
1E isoform in addition to the predominant longer one corresponds to faint but consistent staining in rat cerebellum when transcripts were detected by a riboprobe for the shorter
1E isoform (
Detection of 1E in the Gastrointestinal System
After RT-PCR screening of neuroblastoma cell lines, insulinoma cell lines, and cell lines of the pituitary gland, major expression of an 1Ee isoform was observed in the insulinoma cell lines and subsequently in RNA from the islets of Langerhans (
1E isoforms (Figure 2I), and an MAb against human insulin (Figure 2K). No staining was observed in the negative control when the primary antibody was replaced with preimmune serum (Figure 3I). When antibodies directed against chromogranin A, insulin, and
1E were used, no staining was seen in the exocrine pancreas. In serial sections (Figure 2H2K), the pattern of positive cells in the endocrine pancreas stained by anti-E-spec closely resembled that of chromogranin A used as the marker for neuroendocrine cells. Similar results were obtained with the anti-E-com serum and the purified anti-E serum against an
1E fusion protein, which was recently used for immunohistochemical characterization of
1E in the cerebellum (
1E isoform.
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The immunohistochemical results from the islets of Langerhans led to the hypothesis that the longer 1E isoform may also be expressed in other (neuro)endocrine cells. We used human stomach (antrum mucosa), small intestine, and large intestine (cecum) because the gastrointestinal tract contains more than 20 distinctive endocrine cell types characterized on the basis of their hormonal content (
In the gastric antral mucosa, the immunohistochemical staining pattern and the morphology of chromogranin A-positive cells (Figure 3A) approximately correspond to the cells positively stained with anti-E-spec antibodies (Figure 3B). Anti-gastrin antibodies were used as markers for the G-cells and showed a distribution pattern similar to that seen with anti-E-spec (Figure 3C).
In the mucosa of the small intestine, few dispersed cells were detected by the chromogranin A antibody. These cells were usually located within the epithelium or sometimes within the lamina propria (Figure 3D). A similar picture of scattered positively stained cells was observed with the serum specific for the longer 1E isoforms (Figure 3E and Figure 3F). With the anti-somatostatin antibody, smaller numbers of cells were positive (Figure 3G) compared to the anti-E-spec serum (Figure 3H). Anti-E-spec serum detected groups of positive cells at the bottom of the crypts and single positive cells in other regions (Figure 3H). No staining was detected when the anti-E-spec serum was replaced by preimmune serum or when preincubated with the peptide (see Figure 3I and Figure 3K for negative controls of pancreas and colon, respectively).
In conclusion, anti-E-spec-positive cells are dispersed within the antrum of the stomach, the small intestine, and the large intestine, and appear to co-localize with the chromogranin A-positive neuroendocrine cells. In addition, 1E is also expressed in the Paneth cells and the lamina muscularis mucosae, and therefore may be related to lysozyme secretion or other functions.
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Discussion |
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Transcripts of the 1E voltage-gated Ca2+ channels are detected in rat, rabbit, mouse, and human brain and in other organs from different species (
1E cDNA fragments (
1E isoforms are identified by RT-PCR, which have been described as
1Ed,
1Ee, and
1Ef. The
1Ee isoform is also expressed in rat and human pancreas and kidney (
1E. Specific antagonists of
1E, which react not only in vitro but also in vivo (for SNX-482 see
In our study, the major focus was directed towards the expression pattern of shorter and longer 1E isoforms by using two peptide-directed antibodies. The polyclonal serum anti-E-com was designed to detect all known isoforms, and the anti-E-spec was designed to detect the longer
1E isoforms
1Ed,
1Ee, and
1Ef. Rat cerebellum was investigated because it represents the site of major
1E expression.
The number of peripheral isoforms had been deduced from PCR fragments of each of two regions, the IIIII loop and the carboxy terminus. The estimated structure was confirmed by sequencing subcloned cDNA fragments (1E is expressed. If different isoforms of
1E are related to different functions, then the expression of a single isoform in neuroendocrine cells of the digestive system may help to deduce putative function(s) of this
1Ee isoform.
Expression of 1E Isoforms in the Cerebellum
In human, rat, and rabbit cerebellum, the expression of 1E has been investigated in the past by in situ hybridization (
1E; see
1E (
1E was found by an antisense riboprobe in the cell bodies of cerebellar Purkinje cells and in the granule cell layer (
1E was found to be expressed in Purkinje cells, especially in the cell body extending into the dendritic tree. Axons of Purkinje cells were also stained, but a lower amount of
1E was detected in the molecular layer (
The expression of 1E in Purkinje cells and the granule layer of the cerebellum has been confirmed with peptide-directed antibodies (
1E transcripts in Purkinje cells (
1E transcripts in the granular layer and single Purkinje cells (
1E is close to the limits of detection, whereas the protein density of
1E is sufficient to be detected by antibodies.
By use of common (anti-E-com) and isoform-specific (anti-E-spec) sera in our present study, the expression of longer 1E isoforms was shown in rat cerebellar Purkinje cells, confirming recent reports of
1E expression in human cerebellum (
1E isoforms in the molecular layer. Our data confirm the results of
1E in cerebellum. In addition, we detected positive staining in the molecular layer, which is attributed to a minor expression of shorter isoforms in cerebellum, as can be deduced from a minor amplification of shorter
1E cDNA by RT-PCR (
Immunohistochemical staining of neurons in the granule cell layer was only occasionally noted. Distinct staining of a few neurons in the granule cell layer has been recently reported with another set of peptide specific antibodies (1E in the neurites of cerebellar granule cells in primary culture (
1E isoform is weakly expressed in the granule cell layer.
Expression Patterns of the 1E Ca2+ Channels and the Marker for Neuroendocrine Cells, Chromogranin A, Are Similar in Pancreas and in Cells of the Endocrine Digestive System
Neurons and neuroendocrine cells possess specific organelles for the secretion of peptide hormones, neuropeptides, and neurotransmitters. These signaling molecules are stored either in the small, transparent synaptic vesicles of neurons and their counterparts in endocrine cells or in the so-called large, dense-core vesicles of endocrine cells and their counterparts in neurons (
In selected tissues from the human gastrointestinal tract, a similar, almost identical localization of the longer 1E isoform and chromogranin A has been observed by examination of serial tissue sections of human pancreas, antrum ventriculi, and small and large intestine (cecum). In islets of Langerhans, the number of
1E-positive cells is almost equal to the number of chromogranin A-positive cells but exceeds the number of insulin-positive cells. Therefore, it can be concluded that the longer
1E isoform, probably
1Ee, is co-localized with endocrine cells of the pancreas and with endocrine cells in the gut. This relation of
1E to cells of the neuroendocrine system points to a putative function of
1E-containing Ca2+ channels during secretion of neurohormones and neuropeptides.
Additional staining of Paneth cells and of distinct locations in the lamina muscularis mucosae points to similar or additional functions of 1E that might be related to lysozyme secretion in the gut and to neurotransmitter secretion at the presynaptic nerve terminals (
1A and
1B, the
1E-subunit has been detected in single calyx-type synapses by immunostaining. Therefore, the positive staining detected in the lamina muscularis mucosae might be related to nerve endings rather than to smooth muscle cells.
Voltage-gated Ca2+ Channels and Secretion
The biological processes during neurosecretion and transmitter release include biosynthesis and processing of the propeptide or transmitter, axonal transport of the signal molecules, and Ca2+-dependent secretion from the nerve terminal. The basic mechanisms underlying secretion are assumed to be universal, from yeast to mammalian neurons (
Ca2+-dependent insulin release has been related to several voltage-gated Ca2+ channel types. The dihydropyridine-sensitive 1D and
1C Ca2+ channels initiate major Ca2+ influx (
-conotoxin GVIA, a blocker of
1B/N-type Ca2+ channels (Table 1), does not contribute to the insulin secretion of human B-cells (
Recently, -agatoxin IVA (500 nM), a blocker of
1A/P- and Q-type Ca2+ channels (Table 1), has been shown to inhibit a portion of insulin secretion (
-agatoxin IVA also inhibits recombinant rat
1E Ca2+ channels up to 75%, with an apparent KD of 51 nM (
1E-type Ca2+ channels and that part of the dihydropyridine-resistant insulin release may be guided by
1E Ca2+ channels. During synaptic transmission, Ca2+ influx through P/Q- (
1A), N- (
1B), and R-type Ca2+ channels (
1E) triggers the release of neurotransmitter at a calyx-type terminal in the rat medial nucleus of the trapezoid body with different efficacy (
1A- and
1B-subunits have been localized to two adjacent segments of the intracellular loop connecting Domains II and III. This so-called synaptic protein interaction (synprint) site has been compared with the structurally related
1E-subunit (Figure 4A). It is noteworthy that the Insert I corresponds, in part, to a duplication of the adjacent downstream arginine-rich sequence and is not redundant in the peripheral isoform
1Ee. Overall, this region downstream of Insert I is very divergent in the three structurally related
1-subunits. The low sequence homology of Insert I to
1A or
1B, on the one hand, and, on the other hand, to the sequence homology of the arginine-rich fragment unique in
1E to the peptide recently identified for functional interaction between an L-type Ca2+ channel and the ryanodine receptor (Figure 4B;
1E may be involved in a different mechanism during excitationsecretion compared to
1A and
1B. The identification of in vivo interaction partners would help us to understand the function of
1E in neuroendocrine cells.
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The number of identified Ca2+ channels involved in secretion is increasing, which raises the following question: Why does the endocrine cell need so many similar Ca2+ channels in parallel (1E is that the
1E Ca2+ channels could add an easily modulated Ca2+ channel that is regulated during signaling processes by stimulating and inhibiting cascades as they are realized during secretion (
1E-subunit is modulated in vitro by protein kinase C (
1E might be an important candidate for Ca2+-mediated secretion in addition to L-type Ca2+ channels, and might be modulated by the intercrossing pathways of protein kinase C and somatostatin. The electrophysiological properties of recently identified carcinoid cells of the human gut (
1E Ca2+ channel. In the future, the insulinoma cell line INS-1 or other cell lines that secrete peptide hormones will be used as model systems for investigation of
1E-specific toxins and their effects on hormone secretion.
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Footnotes |
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1 Present address: Institute of Pathology, Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany.
2 Contributed equally to the work.
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Acknowledgments |
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Supported by the Köln Fortune Program/Faculty of Medicine, University of Cologne (to MW and TS) and by the Center of Molecular Medicine Cologne/Zentrum für Molekularbiologische Medizin Köln (Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Förderkennzeichen 01 KS 9502, to TS and JH).
We thank Dr W. Nastainczyk (Universität des Saarlandes, Homburg) for the synthesis of peptides and antibodies, Ms M. Chludek (Pathologie/Leverkusen) for her permanent help during preparation of serial sections, and Ms R. Clemens, Ms S. Schulze, and Ms U. Tampier for technical assistance.
Received for publication December 9, 1998; accepted April 2, 1999.
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