ARTICLE |
Correspondence to:
Toni Schneider, University of Köln, Institute of Neurophysiology, Robert-Koch-Str. 39, D-50931 Köln, Germany. E-mail:
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Summary |
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The calcium channel 1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of
1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac
1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to
1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of
1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of
1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of
1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of
1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca2+-dependent secretion of peptide hormones such as ANP and urodilatin. (J Histochem Cytochem 48:807819, 2000)
Key Words:
1E calcium channels, R-type Ca2+ channel, endocrine, chromogranin, atrial natriuretic peptide, urodilatin, H9c2 cells, conducting system, blood pressure regulation
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Introduction |
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Cardiomyocytes express T- and L-type voltage-gated Ca2+ channels (1C-containing L-type Ca2+ channel by gene targeting is lethal before birth (
1C,
1D has also been detected by antibodies in functional studies of guinea pig heart (
Using electrophysiological methods, cardiac T-type Ca2+ channels were detected not only in sinoatrial node cells (
Three new T-type voltage-gated Ca2+ channels (1G,
1H, and
1I) have recently been cloned and expressed in recombinant systems (
The detection by Northern blotting of two of the three cloned low-voltage-activated Ca2+ channel subunits (1G,
1H) in heart (
1E-induced Ca2+ inward currents, because antisense oligonucleotides against
1E mRNA inhibited the IGF-induced T-type current (
1E is not considered a typical T-type current (
1E as a method independent of RT-PCR amplification (
1E in heart. Therefore, the distribution of
1E was compared with the expression of neuroendocrine markers (chromogranin A/B) in the myocardium. Furthermore, the distribution of
1E was investigated in human kidney because this is another organ that contains natriuretic peptides and plays an important role in blood pressure regulation.
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Materials and Methods |
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RNA isolation and RT-PCR
Total RNA was isolated from fresh or from shock-frozen rat brain (cerebrum and cerebellum) or from rat heart using standard protocols (
Fragments of cDNA were amplified by PCR (PerkinElmer Cetus Instruments; Norwalk, CT) after reverse transcription of total RNA using standard protocols as specified (1 subunits from high-voltage-activated Ca2+ channels and for the housekeeping enzyme hypoxanthine phosphoribosyltransferase have been summarized recently (
1G cDNA fragment was amplified using the primer Gfrwsp, 5'-AGCCCCGGTGGTTTCTTCTA-3' (nt 650670) of rat
1G cDNA sequence (GenBank
AF027984; Bethesda, MD) as forward and Grevsp, 5'-TGAGCGGTCGCAGCACAC-3' as reverse primer (nt 10471030). The rat
1H cDNA fragment was amplified using oligo-452341, 5'-AGGAGGCTC-GGCGCCGG-3' (homologue to human
1H, AF051946, nt 47774793) as forward and oligo-452340, 5'-GGATAGGAGGACGATGGCCAA-3' (homologue to human
1H, nt 51315150) as reverse primer. The rat
1I cDNA fragment was amplified using Ifrwis, 5'-GCTGCGGCGCCTGGAAAAGAA-3' (nt 45014521) of rat
1I cDNA sequence (GenBank
AF086827) as forward and Irevis, 5'-GCCCATGCACGGACAGCAGCACAAT-3' as reverse primer (nt 48384814). The annealing temperatures were 57C for
1G and
1H, and 60C for
1I cDNA fragments.
Oligonucleotide primers were purchased from Eurogentec Bel SA (Seraing, Belgium) or from Gibco/Life Science (OligoGold; Gaithersburg, MD).
Subcloning and Sequencing
Amplified cDNA fragments of critical regions were subcloned into the pCR2.1 TOPO vector according to the recommendations of the manufacturers and including the TOPO TA cloning kit (Invitrogen; Leek, The Netherlands). Plasmid DNA was sequenced according to the method of
Antibody Production
Two peptides homologous to human 1E sequences were coupled to hemocyanin and used for immunization of rabbits. Peptide Nast-195 corresponds to a common sequence in all published
1E isoforms and peptide Nast-197 is part of a 43-amino-acid extension in the carboxy terminus of the fetal brain-derived
1Ed isoform (
1E isoforms,
1Ee and
1Ef (
1E sequence. Peptide Nast-197 contains one additional cysteine residue for coupling to a matrix, as shown in brackets. Immunization of the rabbits with the hemocyanin-coupled peptides was described previously (
The sensitivity and specificity of the anti-1E sera were tested by immunoblots of microsomal membrane proteins from stably transfected HEK-293 cells, which express the human
1Ed (cell line HEK-2C6 or HEK-2C2), or the deletion mutant
1Ed-CDEL (cell line HEK-
1E-CDEL; see
Preparation of Paraffin-embedded Tissue and Immunostaining of Tissues and H9c2 Cells
The procedures used in the present study that involved tissues from animals and human were performed in accordance with the regulations governing their use in scientific research.
Female Wistar rats weighing 180250 g were anesthetized with CO2, decapitated, and the organs rapidly removed. Tissue sections from human kidney were obtained from surgically resected specimens. All tissues were fixed in 10% PBSbuffered formalin at room temperature overnight and embedded in paraffin (
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 putting the slides in 0.3% hydrogen peroxide in methanol for 20 min at 37C. After washing with PBS, sections were immunostained by the streptavidinbiotinhorseradish peroxidase technique (1E.
In some experiments, the purified anti-1E serum against an
1E fusion protein (
1E, a gift from Dr. R.E. Beattie and Dr. S.G. Volsen (E. Lilly; Windlesham, UK), were used as additional antibodies to compare the staining results with different primary antibodies.
Isolation of Membrane Proteins and Western Blot Analysis
Stably transfected HEK-293 cells were grown as described (
The membrane proteins were separated by polyacrylamide gel electrophoresis according to standard protocols and immunoblotting was performed as reported (1E-spec serum was not observed with the shorter splice variant
1E-CDEL (
1G-,
1H-, or
1I-transfected cell lines.
Protein concentrations were determined with the BCA method (Pierce; Rockford, IL). The specificity of the sera was also tested by immunostaining untransfected and stably transfected HEK-293 cells expressing the cloned human 1E (
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Results |
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Transcripts of Voltage-gated Ca2+ Channels in Rat Heart
Total RNA was extracted from freshly isolated rat heart and reverse transcribed. Oligonucleotide primer pairs were used to amplify eight different 1 subunits either from rat cerebrum as a reference or from rat heart cDNA (Fig 1). The transcripts of two L-type
1 subunits,
1C and
1D, were detected. In addition, fragments of two non-L-type (
1A and 1E) and two T-type subunits (
1G and
1H) were amplified (Fig 1). In the cardiac rat cell line H9c2, neither
1D,
1A, nor
1H was observed, but low amounts of
1I were detected (Fig 1). For
1C and
1H, additional splice variants were deduced by the size of the amplified fragment. No transcripts were amplified with primer pairs for
1S in adult rat heart (not shown).
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Splice Variant of 1E Voltage-gated Ca2+ Channel in Heart
Previous studies using RNA extracted from human frozen tissue failed to detect the presence of 1E transcripts (
1E transcripts were detected extracting total RNA from rat cardiac tissues immediately after dissection without prior freezing (Fig 2). However, the transcripts of
1E were still amplified in much lower amounts than from the same amount of total RNA of rat cerebellum (Fig 2A and Fig 2B). The structure of the cardiac
1E splice variant was deduced by amplifying indicative cDNA fragments of the linker between Domains II and III (II/III loop; see Fig 2A) and the carboxy terminus (Fig 2B). After subcloning and sequencing of fragments from the II/III loop (Fig 2C), the comparison of the
1E fragment from rat heart showed 100% identity with the cloned rat neuronal and 92% identity with the human neuronal
1E amino acid sequence when the aligned sequences between insert 1 and insert 2 (Fig 2C) were compared. Insert 1 was absent in the
1E splice variant detected in heart, whereas insert 2 was present as in the neuronal
1E. However, the amino acid sequence within insert 2 was highly divergent within its seven amino acids compared to the human
1E (Fig 2C). Sizes and sequences of the cDNA fragments from both the II/III loop and the carboxy terminus led to the conclusion that the same
1Ee splice variant is expressed in rat heart as in rat islets of Langerhans and human kidney (
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Specificity of the Anti-1E Serum
After immunoblotting of microsomal membrane proteins, no positive staining was detected in proteins from untransfected HEK-293 cells, whereas in membrane proteins from HEK-2C6 cells a protein of 243 ± 22 kD (n = 4) (Fig 3A) was observed. The apparent size is smaller than the predicted size of the cloned 1Ed (261,766 Daltons).
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The immunodetection of 1E was dependent on the temperature during SDS-PAGE and on the pretreatment of the membrane proteins before electrophoresis (Fig 3B). When the temperature during electrophoresis was decreased to 10C, the apparent staining of the anti-
1E-positive band was more intense than after electrophoresis at 22C (Fig 3B; compare Lanes 24 with Lanes 68). Denaturing of the protein aliquots by SDS denaturing buffer before freezing also diminished the intensity of staining (Fig 3B, Lanes 6 and 8), leading to the conclusion that either the overall integrity of
1E or the renaturation of the anti-
1E-spec epitope after denaturing electrophoresis is sensitive to elevated temperature.
The detection of single protein bands in microsomal membranes from stably transfected cells suggests that the polyclonal sera are highly specific for the corresponding 1 subunit. The reactivity and the specificity of the polyclonal anti-
1E-spec serum had been evaluated recently by immunocytochemistry (
1E splice variants in cell lines and tissues.
Immunoblotting of Microsomal Membrane Proteins from Rat and Guinea Pig Tissues
Microsomal membrane proteins were isolated from rat and guinea pig heart, and from rat cerebrum, the rat insulinoma cell line INS-1, and rat kidney. The solubilized membrane proteins were immunoblotted after PAGE. The anti-1E-spec serum stained a protein of 218 ± 6 kD (n = 3) in the cardiac microsomes from both species (Fig 4A). The size of the positive band in rat and guinea pig heart was smaller by 28 kD than the recombinant protein from the stably transfected HEK-293 cell lines. Microsomes of rat cerebrum were used as a reference tissue, because a different
1E isoform is expressed lacking the anti-
1E-spec epitope (
1E-spec serum (Fig 4A, Lane 7). However, in INS-1 cells and in kidney, the anti-
1E-spec serum stained a protein of similar size as in HEK-2C6 cells (Fig 4A). In addition, a shorter positive band was also observed in rat kidney membrane proteins.
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The second anti-1E serum designed to detect all known
1E isoforms also stained a 218-kD protein from rat and guinea pig heart (Fig 4B). In addition, a longer
1E was observed in the heart from guinea pig and in rat cerebrum (Fig 4B). No staining was detected when preimmune serum was used instead of primary antibody.
In rat heart, the molecular size of the putative cardiac 1E was smaller than the size of the recombinant protein after staining with both anti-
1E sera (see arrows in Fig 4A and Fig 4B). On the basis of RT-PCR results, a cardiac-specific
1E splice variant should be truncated by only 2.5 kD in the II/III loop. However, the observed shift was 28 kD and was in the same range as detected for other recombinant
1 subunits of voltage-gated Ca2+ channels in purified skeletal muscle, cardiac muscle, and neurons (
1Ee isoform, which is structurally different from the major neuronal
1E isoform, was detected in rat and guinea pig heart as well as in rat INS-1 cells and human kidney, but not in rat cerebrum.
Immunohistochemical Detection of 1E in Heart
The 1E splice variant detected in rat and guinea pig heart by RT-PCR and by immunoblotting was predicted to contain the longer carboxy terminus that was originally cloned from human fetal brain (
1E-spec serum throughout this part of the report (Fig 5) but identical results were obtained with the anti-
1E-com serum and an anti-
1E serum used for the detection of neuronal
1E (
1E-spec-positive cells were detected and were scattered throughout the rat heart, in the right atrium (Fig 5A), the left atrium, (Fig 5F), and in both ventricles (Fig 5A and Fig 5B). No staining was observed with the preimmune (Fig 5C and Fig 5D) or the preabsorbed serum (not shown).
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To discriminate the conducting muscle fibers from the force-producing muscle fibers, we evaluated hematoxylineosin-stained tissue sections (Fig 5E). The cytoplasm of the conducting muscle fibers was less eosinophilic and therefore appeared paler. In addition, it often contained vacuoles or appeared to be "empty," probably due to the fact that conducting muscle fibers have a lower content of regularly organized contractile material and increased numbers of glycogen granules (
Only the subpopulation of cardiac muscle cells histochemically defined as impulse-conducting fibers (Fig 5E) were anti-1E-spec-positive (Fig 5F). A similar staining pattern was observed in serial sections of the right atrium and both ventricles (not shown). No staining was detected with preimmune serum (Fig 5G) or peptide-blocked sera (not shown). Neither preincubation of the sera with 20 µM hemocyanin, the carrier protein for immunization with peptides, nor preincubation with 20 µM cardiac myosin, a major protein of similar size associated with microsomal membranes, blocked the anti-
1E-spec-positive staining pattern observed in rat heart. Therefore, in rat heart
1E-expression appears to be restricted to the conducting fibers of the myocardium, including the bundles of His and the Purkinje fibers of the ventricles.
In serial tissue sections, anti-chromogranin A/B (Fig 6A) and anti-ANP antibodies (Fig 6C) were used as markers for endocrine cells, first because we had described 1E in neuroendocrine cells of the gastrointestinal system (
1E-spec (Fig 6B). Atrial (Fig 6C) but not ventricular muscle cells (not shown) were stained by the anti-ANP serum. No positive staining was seen when preimmune serum for
1E was used instead of primary antibody (Fig 6D). We conclude that
1E is co-expressed with chromogranin A/B in the rat heart and with ANP in the rat atrium.
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In cross-sections through the bundle of His (truncus atrioventricularis), bundles of conducting fibers (Fig 6E, central part) reacted strongly with the anti-1E-spec serum. The immunohistochemically defined distribution of
1E led us to suggest that it may be involved in both endocrine and conducting functions in rat heart.
To confine and to confirm the expression of 1E in muscle cells related to the endocrine system of the rat heart, expression of the
1E subunit (Fig 7B and Fig 7C), ANP (Fig 7A), and chromogranin (Fig 7D and Fig 7E) was investigated in the rat cardiomyoblast cell line H9c2. These cells were positively stained by anti-
1E-spec, anti-ANP, and anti-chromogranin, but not by the preimmune serum (Fig 7F).
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Immunohistochemical Detection of 1E in Kidney
The secretion of the peptide hormone urodilatin in kidney serves similar functions as the ANP secretion in heart (1E-spec serum revealed a strong positive reaction in distal tubules and a weaker one in proximal tubules (Fig 7G). No staining was observed in the glomerulus (Fig 7G) or in the negative control incubated with preimmune serum (Fig 7H). Identical patterns were observed with the anti-
1E-com or the serum received from Dr. S.G. Volsen (
In summary, 1E was detected by RT-PCR in rat and human kidney (
1E might be related to functions typical for the distal tubules in kidney.
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Discussion |
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Transcripts of Voltage-gated Ca2+ Channels in Rat Heart
In heart, T- and L-type voltage-gated Ca2+ channels are involved in pacemaker activity and excitationcontraction coupling. After the reported cloning of 10 different 1 subunits, a more detailed analysis by RT-PCR reveals that at least transcripts of two different L-type (
1C,
1D), two non-L-type (
1A,
1Ee), and two T-type Ca2+ channels (
1G,
1H) are amplified from rat heart. In the present study, one of them, a specific splice variant of
1E, was characterized in greater detail. The expression of
1E in rat and guinea pig heart was also investigated at the protein level because an IGF-induced T-type Ca2+ current was reduced in rat atrial myocytes after application of
1E-specific antisense oligonucleotides (
Initially, we failed to amplify 1E transcripts from frozen human heart (
1E. The most indicative cDNA fragments (II/III loop) were subcloned and identified by sequencing to be related to the same
1Ee isoform as found in islets of Langerhans and kidney (
The 1Ee isoform is found in neuroendocrine tissues and is also expressed in cerebellum. Its tissue-specific expression in islets of Langerhans, endocrine cells of the gastrointestinal tract (
1E within endocrine signaling pathways.
The primary structure of the cardiac 1Ee isoform shows major differences from the cloned neuronal
1Ed (
1E-3 (
1Ee and starts at position aa 749 with RERRRRHHMS, resembling the adjacent sequence at position aa 768 of human
1E, RERRRRHHMS. On the other side, this sequence shows homology to a skeletal muscle
1S sequence, EERKRR.KMS, which is also located in the II/III loop of
1S and is believed to be involved in excitationcontraction coupling (
1E during excitationsecretion coupling, as was shown for
1A,
1B (
1C (
The carboxy terminus of 1Ed and
1Ee contains an insertion of 43 amino acids of yet unknown function. After deletion of these 43 amino acids, the basic electrophysical properties are not changed in the deletion mutant compared to the wild-type
1Ed, as was shown after stable transfection in HEK293 cells (
Detection of 1E by Immunoblotting
The apparent molecular size of 1E was determined by immunoblotting of microsomal membrane proteins from several tissues, including rat and guinea pig heart. The cardiac protein, which was stained by two different anti-
1E sera after immunoblotting, is truncated to some extent and its apparent size is smaller by 28 ± 16 kD than the reference protein. A shift of only 2.5 kD was expected because the recombinant
1Ed contains the 19-aa insert 1 of the II/III loop. Because both sera detected
1E in rat and guinea pig heart, the
1E subunit might be truncated downstream of the epitope recognized by anti-
1E-spec. The truncation might be caused either by posttranslational processing or by in vitro proteolysis during isolation of the membrane proteins, or by both.
Detection of 1E in Fibers Related to the Impulse-conducting System of the Rat Heart and in the Distal Tubules of Human Kidney
Three different sera against 1E were used during the immunohistochemical investigation of anti-
1E positive muscle cells in the rat heart. Two sera were created by linking peptides to hemocyanin (
1E-directed sera. Anti-
1E-positive muscle cells were found in the right and left atrium and also in both ventricles. The cell morphology itself and the HE staining of Purkinje fibers clearly demonstrated that the anti-
1E-positive muscle cells are part of the impulse-conducting system.
Several findings support our interpretation of an endocrine function for 1E in rat heart. First, the distribution of anti-
1E-positive cells in heart closely resembles the distribution of chromogranin A/B, which is co-localized with ANP (
1E in atrium as well as in ventricle of rat and guinea pig heart might participate in triggering the secretion of peptide hormones, comparable to its recently deduced function in islets of Langerhans and endocrine cells of the gastrointestinal system (
Before the cardiac expression of 1E had been investigated, its distribution in human kidney had been analyzed. The fact that anti-
1E-positive cells were found mainly in the distal tubules could also be related to an endocrine function of
1E for the secretion of urodilatin in kidney. The renal natriuretic peptide urodilatin is synthesized in the distal tubule region (
Other functions, such as impulse generation and pacemaking, might also be related to the increasing number of putative T-type Ca2+ channels identified in heart. Recent reports have shown that T-type Ca2+ currents not only contribute to pacemaking (1E-specific peptide toxins (
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Footnotes |
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1 These authors 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 Köln, and the Center of Molecular Medicine Köln/Zentrum für Molekulare Medizin Köln (Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Förderkennzeichen 01 KS 9502).
We thank Prof Dr J. Hescheler for providing us his help and excellent opportunities in his facilities for research, and Dr R.E. Beattie, Dr P.J. Craig, and Dr S.G. Volsen (Lilly Company; Windlesham, London) for a third serum against 1E. We acknowledge the careful reading of the manuscript by Prof Dr E. PerezReyes (Charlottesville, VA). We thank Ms M. Chludek (Leverkusen, Germany) for her help during preparation of serial sections, and Ms R. Clemens and Ms S. Schulze for their permanent technical assistance.
Received for publication February 4, 2000; accepted February 9, 2000.
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