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Ca2+Exchanger in the Guinea Pig Cochlea
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
233, 737–741 (1997)
RC976533
Alternatively Spliced Isoforms of the Na//Ca2/ Exchanger in the Guinea Pig Cochlea Takeshi Oshima,1 Katsuhisa Ikeda, Masayuki Furukawa, and Tomonori Takasaka Department of Otolaryngology, Tohoku University School of Medicine 1-1, Seiryo-machi, Aoba-ku, Sendai 980-77, Japan
Received March 17, 1997
The cochlea has been suggested to express some Na// Ca2/ exchangers (NCX), since efficient acoustic transduction requires cytosolic calcium homeostasis. The present study revealed that several spliced isoforms of NCX are expressed in the guinea pig cochlea. Moreover, to determine their localization in the cochlea, microdissected RT-PCR was performed. The guinea pig cochlea was microdissected into three parts (lateral wall, the organ of Corti and modiolus). The cochlear lateral wall and the organ of Corti expressed only a single isoform of NCX1. On the other hand, five isoforms of NCX1 and four isoforms of NCX3 were detected in the cochlear modiolus. The alternative splicing may provide diverse functions for NCX in the cochlea. q 1997 Academic Press
The sodium-calcium exchanger (NCX) is an electrogenic transporter protein expressed broadly in animal cells. It is well characterized in excitable tissues, such as the heart, and mediates the exchange of one intracellular calcium ion for three extracellular sodium ions. In cardiac muscle, NCX is responsible for removing almost all of the calcium that enters via the calcium current (1). The NCX has also been shown to be present in particularly high concentrations in the neuronal synapses (2) and its mRNA is detected in the brain tissues (3) where it may play a critical role in the modulation of neuronal transmission (4). Besides these electrically excitable cells, kidney possesses the NCX which plays an important role in the regulation of Ca2/ reabsorption (5). The functional diversity of NCX in these tissues probably results from the tissue-specific expression of NCX. Up to the present, three isoforms, NCX1, NCX2 and NCX3, which are transcribed from three distinct genes, have been identified (6,7,8). Several tissue-specific isoforms of the NCX1 are known to result from alternative splicing in the long putative intracellular loop region (9). Distinct isoforms are expressed in 1
Correspondence to Takeshi Oshima. Fax: 81-22-717-7307.
heart, kidney and brain (9). NCX2 is detected only in brain and skeletal muscle (7). NCX3 is expressed in brain and skeletal muscle (8). The cochlea, which is the sound-perceptive organ, contains three functionally distinct tissues (the organ of Corti, the spiral ganglion and the stria vascularis). The organ of Corti possesses two types of hair cells both of which are auditory sensory receptors. The stria vascularis is the non-sensory ion-transporting epithelial tissue the characteristics of which are similar to those of kidney. The spiral ganglion cells are neuronal cells that transduct the sound stimulus to the brain. It is possible that NCX isoforms are expressed heterogeneously, since the cochlea is a complicated organ. Although we have previously suggested the presence of a Na//Ca2/ exchanger on the plasma membrane of a cochlear outer hair cell by electrophysiological study (10), it has not yet been determined which isoforms are expressed. The aim of the present study is to verify the presence of the NCX molecules in the guinea pig cochlea by RTPCR techniques. Moreover, their localization in the cochlea was investigated by RT-PCR, since it has been demonstrated that alternative splicing results in the presence of several tissue-specific isoforms of the NCX1 in the cardiac muscle, kidney and brain (9). The alternatively spliced exons are too short in size (the shortest is comprised of 15 nucleotides) to enable us to investigate the localization of the isoforms by immunohistochemistry and ISH. Rather, we applied RT-PCR as successfully performed in the kidney (11) to the microdissected cochlear segments, since RT-PCR for cochlear tissues has been established (12-17). We could reveal the distribution of the spliced isoforms of NCX in the three parts of the guinea pig cochlea. EXPERIMENTAL PROCEDURES cDNA synthesis and polymerase chain reaction from the whole cochlea. Young adult guinea pigs were anesthetized with diethylether and then sacrificed by decapitation. Their temporal bones were removed and the bony capsules were picked away in autoclaved phos-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS shown in Fig. 3B, were used on another nested amplification. The conditions of PCR were the same as mentioned above. To confirm the cochlear microdissection morphologically, the specimens were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The organ of Corti was mounted on a glassslide and then overlaid with a glass coverslip, followed by examination under a light microscope. The cochlear lateral wall and decalcified modiolus were postfixed with 1% osmium tetraoxide, dehydrated, and embedded in Epon 812. The samples were examined under a light microscope after thin sectioning and staining with toluidine blue.
FIG. 1. Primers. Four degenerate primers (f1, f2, r1 and r2) were synthesized based on the sequences of rat NCX1 and human NCX2. The guinea pig NCX1-specific primers (f3 and r3) were synthesized based on the sequences obtained in the present study and the guinea pig cardiac NCX. The forward and reverse primers (f4 and r4) were derived from the sequence obtained in this study (Fig.3B). Bases in parentheses indicate degeneracy.
phate-buffered saline under a stereomicroscope. Polyadenylated RNA was isolated from the whole cochlea using a mRNA isolation kit (Quick Prep Micro mRNA Purification Kit; Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions. The firststrand cDNA was synthesized from the mRNA with a commercially available kit (First-Strand cDNA Synthesis Kit; Pharmacia), in which a random hexanucleotide-primer and Molony murine leukemia virus reverse transcriptase were used. Fifteen ml of first-strand cDNA solution were obtained from two cochleas. One ml of the aliquot was used as a template for PCR. To amplify cDNA molecules of the guinea pig NCX members by RT-PCR, four degenerate primers (Fig. 1) were synthesized based on the sequences of canine (6), rat (18), and rabbit (9) NCX1 and human NCX2 (7). The forward primers from and f2 recognize the nucleotides encoding amino acids 478-486 and 530-537, and the reverse primers r1 and r2 (antisense) the amino acids 724-731 and 710-717 in the canine NCX1. Amino acid sequences in these regions are completely identical among the exchangers of the four species. The first-strand cDNA from the guinea pig cochlea and the sense and antisense primers f1 and r1 were used for RT-PCR. Thereafter, the resulting cDNA was amplified by the nested PCR with the internal primers f2 and r2. PCR was performed with a programmable thermal cycler (Model 480; Perkin Elmer, Norwalk, CT) and a thermostable Taq DNA polymerase (TaKaRa Taq; Takara Shuzo, Otsu, Japan). The initial PCR was repeated for 30 cycles as follows: 947C for 60 s, 557C for 120 s, and 727C for 120 s. The second PCR was performed under the following conditions: 947C for 45 s, 587C for 45 s, and 727C for 60 s, for 40 cycles. The procedure was completed by continuation of the last 727C incubation for a further 7 min. PCR products were analyzed by electrophoresis on 2-3 % agarose (SepRateTM-SDF, Amersham, Buckinghamshire, UK) gels.
RESULTS Whole cochlea study. PCR amplification using the guinea pig cochlear cDNA as a template was performed. The 40-cycle amplification with the primers f1 and r1 produced a faint band on an agarose gel (data not shown). To facilitate cloning the PCR product, we decided to run nested PCR with the primers f2 and r2 and the primary PCR product as a template. Agarose gel electrophoresis of the nested PCR product revealed a clear, broad band of 400-500 bp (Fig. 2). The broadness of the band suggests the heterogeneity of the PCR product. This band was not detected when the guinea pig genomic DNA was PCR-amplified under the same conditions (data not shown), indicating that this product was not derived from the genomic DNA. We performed sequencing of the fragments to confirm the heterogeneity. The PCR products were ligated to pCR II and then transformed. Hundreds of colonies were obtained. Thirty of them (gpncx1-30) were picked for plasmid isolation and restriction analysis, which revealed that 23 clones contained inserts. They were sequenced and all of them comprised open reading frames. Amino acid sequences of 7 clones were highly homologous to various spliced variants of the rabbit NCX1 (Fig.3A). The amino acid sequences of the other 16 clones were not homologous to NCX1. The sequence of the cDNA clones, gpncx8 and 12, was highly homologous to the rat NCX3 sequence (Fig.3B). The amino acid identity in this region was 97.0%. Moreover, we found three
Sequencing. The PCR products were TA-cloned into pCRTMII vector (InVitrogen, San Diego, CA). Purified plasmid DNA was sequenced bidirectionally using a fluorescent sequencing system (ALFexpress, Pharmacia). Cochlear microdissection and NCX cDNA amplification. After removing the bony cochlear wall, the lateral wall of the cochlea was peeled and collected by using fine forceps under a stereomicroscope. Next, the organ of Corti was stripped and put into a tube by the forceps. The cochlear modiolus, in which the lateral wall and the organ of Corti were excluded, was collected into a different tube. For RT-PCR, first strand cDNA was synthesized as described above. After the initial PCR with the primers f1 and r1, the nested PCR was performed with the NCX1-specific internal primers f3 and r3 (Fig. 1), based on the sequences obtained in the present study using the whole cochlea and the guinea pig cardiac NCX (19). Simultaneously, primers f4 and r4, which were derived from the sequence
FIG. 2. RT-PCR amplification of NCX in guinea pig cochlea. The nested PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide (right lane). Marker: fX174 digested with HaeIII (left lane).
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Morphological findings of cochlear microdissection. Guinea pig cochleas could be divided into three parts: 1) the cochlear lateral wall, 2) the organ of Corti and 3) the modiolus. These microdissected specimens were examined under a light microscope, which could disclose no cross-contamination. The stria vascularis, spiral ligament and spiral prominence were included in the specimen of the lateral wall (Fig.4A). The dissected Corti’s organ was observed in the Nomarski image, in which outer hair cells (OHCs) and lipid drops in Hensen’s cells could be clearly seen (Fig.4B). Deiters’ cells and pillar cells were also probably included in the specimen of the organ of Corti. In the specimen of the modiolus, the main cell-contents were spiral ganglion cells (Fig.4C). Vessels in the modiolus were observed. Few cells of the organ of Corti could be seen. Although a large amount of efferent nerve fibers were present, these fibers do not potentially contribute to RT-PCR due to the absence of ribosomes in axons.
FIG. 3. Sequencing analysis of NCX in the guinea pig cochlea. Twenty-three clones from the whole cochlea were sequenced. Polypeptide sequences are given in single-letter codes. A: Four distinct sequences were obtained from 7 clones (gpncx2, 4, 9, 16, 18, 23 and 24). Their sequences were highly homologous to rat and rabbit NCX1 sequences. Amino acids that differ from the published rat sequence are indicated by underlines. As in rabbit and rat NCX1, they were constructed by constitutive upstream and downstream sequences and alternatively spliced exons A, B, C, D and E. Their exons were named after genomic DNA sequences of rabbit NCX1 (9). The isoform NACA6 was described in rabbit brain (9). The NACA8, NACA9 and NACA10 have been identified in this study. Dashes represent gaps that were introduced in the sequences to optimize alignment. Vertical lines denote boundaries of exons. B: The amino acid sequence was obtained from 2 clones (gpncx8 and gpncx12). This was highly homologous to rat NCX3. Identities are indicated by asterisks. In 2 clones (gpncx6 and gpncx15), the underlined sequence was replaced as a possible alternative spliced exon by the sequence as follows: VHIKVIDDEAYEKNKNYFIEMMGPRMVDMSFQK. Sequencing of the other 11 clones revealed that 6 amino acids indicated by a double underline were deleted. In a clone (gpncx17), the amino acids double underlined was deleted from the sequence of gpncx6 and gpncx15. The primers f3, r3, f4 and r4 recognize the nucleotides encoding amino acids enclosed by boxes.
other cDNA isoforms due to deletion or replacement. Sequencing 11 clones revealed that 6 amino acids (ALLLSP) were deleted from the sequence shown in Fig.3B. In the other 3 clones (gpncx6, 15 and 17), the underlined sequence in Fig.3B was replaced by the sequence: VHIKVIDDEAYEKNKNYFIEMMGPRMVDMSFQK. Moreover, the 6 amino acids (ALLLSP) deletion took place in the clone gpncx17.
FIG. 4. Microdissection of guinea pig cochlea. Panel A: Stria vascularis (arrow 1), spiral ligament (arrow 2) and spiral prominence (arrow 3) are observed in the segment of the cochlear lateral wall. The lateral part of the basilar membrane (arrow 4) is seen but the organ of Corti is excluded. Panel B: On the Nomarski view, Hensen’s cells (arrow 5) and outer hair cells (arrow 6) can be easily identified in the segment of the organ of Corti. Panel C: The cochlear modiolus, in which the lateral wall and the organ of Corti were excluded, mainly contains spiral ganglion cells (arrows 7) and nerve fibers (arrow 8). BarÅ100 mm.
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FIG. 5. PCR products generated from three microdissected regions of guinea pig cochlea. Lanes 1–3: nested PCR with the primers f3 and r3. A single band of approximately 400 bp was detected in the samples of both the cochlear lateral wall (lane 1) and the organ of Corti (lane 2). On the other hand, the heterogeneous fragments were examined at a slightly higher position in the cochlear modiolus sample (lane 3). Lanes 4–6: the primers f4 and r4 were used for the nested amplification. The principal product amplified from modiolus cDNA is observed as two bands approximately 390 bp long (lane 6). This heterogeneity might be derived from alternative splicing. In lane 4 (lateral wall) or lane 5 (the organ of Corti), no band of the expected size is seen. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Markers were fX174 digested with HincII (lanes M ).
Localization of NCX1 isoforms in cochlea. To examine the localization of NCX1 molecules among the three cochlear parts, the nested PCR was performed with the nested primers f3 and r3. The PCR products generated from the three microdissected regions were first checked by agarose gel electrophoresis (Fig. 5). The amplified products from the lateral wall and the organ of Corti were detected as a single band. A relatively broad band at a position higher than those from the lateral wall and the organ of Corti was observed in the sample of the modiolus. Sequencing analysis was next performed. In all the clones from the lateral wall (nÅ8) and the organ of Corti (nÅ5) the exons B and D were expressed, whereas the other exons A, C and E, were excluded. Five isoforms, of which the exon patterns were ACDE (nÅ4), AD (nÅ2), ADE (nÅ1), BD (nÅ2) and BDE (nÅ3), were found by sequencing 12 clones from the modiolus. Localization of putative NCX3 in cochlea. A sequence highly homologous to rat NCX3 was detected in the guinea pig cochlea by RT-PCR in this study. The cochlear localization of this molecule was investigated by RT-PCR with the microdissected cochlear tissues. The nested PCR with the primers f4 and r4 produced a band of the expected size from the modiolar tissue, but not from the cochlear lateral wall or the organ of Corti. DISCUSSION This study revealed that multiple isoforms of NCX were expressed in the guinea pig cochlea. It is considered that NCX protein has 11 putative transmembrane domains and a long intracellular loop domain between
the 5th and 6th transmembrane domains (20). In this intracellular loop, alternative splicing takes place. The rabbit (9) and rat (21) NCX1 has six alternatively spliced exons designated as A-F in this region. Our sequencing analysis of this region demonstrated that the exons of the guinea pig NCX1 are almost identical to those of the rabbit and rat NCX1. We found five exons corresponding to exons A-E, however, exon F could not be detected in this study. This result does not indicate a lack of exon F in the guinea pig NCX1, since the guinea pig cardiac NCX1 possesses exon F (19). Rather, alternative splicing might possibly exclude exon F in the guinea pig cochlea. In this study, the expressed exon patterns of seven whole cochlear clones were ACDE (nÅ3), ADE (nÅ2), ACD (nÅ1) and BDE (nÅ1). The pattern ACD (the clone gpncx9) has been described as NACA6, which is expressed in the brain (9). Other patterns, ACDE, ADE and BDE, which have never appeared in other tissues or species, corresponded to novel spliced variants. We designated them as NACA8, NACA9, and NACA10, respectively. We could detect the clones gpncx8 and gpncx12 which were not highly homologous to NCX1. Rather, they were similar to rat NCX3. Since the amino acid identity in this region was 97.0%, this sequence is presumed to be guinea pig NCX3. We report here 14 other clones which are considered to be spliced variants of gpncx8 and gpncx12. No sequences homologous to human NCX2 were found in this study. The RT-PCR following the cochlear microdissection revealed the cochlear localization of spliced isoforms of NCX1 and NCX3. NCX3 messages could be detected only in the modiolus. Spiral ganglion cells in the modiolus possibly express NCX3 since high levels of it are expressed in brain (8). With respect to NCX1, the expressed exon pattern BD was detected in all three cochlear parts. This isoform is described as NACA3, which is expressed predominantly in kidney (22). It is additionally expressed in eye, aorta, intestine and thymus (23). The expressed exon patterns in the modiolus were ACDE (NACA8), AD, ADE (NACA9) and BDE (NACA10), besides BD (NACA3). The isoform AD, which was first isolated from rat brain (24), has been described as NACA4 (9). NACA6 obtained from the whole cochlea (gpncx9) could not be detected by the microdissected cochlear RT-PCR. This might be rarely expressed in cochlea. PCR products obtained from the whole cochlea seemed to be derived from the modiolus, not from the lateral wall or the organ of Corti. This result may be explained by the fact that the number of cells in the modiolus is greater than those in the lateral wall and the organ of Corti. Whole cochlear RTPCR is unlikely to reflect messages derived from specific cochlear regions such as the stria vascularis and the organ of Corti. Therefore, microdissected rather than whole tissue RT-PCR in the cochlea is useful to investigate the localization of messages.
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We did not demonstrate the cellular distribution of NCX in the cochlea, however, immunohistochemical and physiological investigations have given us some information on the cochlear localization of NCX. Na// Ca2/ exchange activity has been detected in cochlear outer hair cells using fluorescent probes (10). A monoclonal antibody against NCX1 has recognized strial marginal cells and Hensen’s cells (25). Our RT-PCR demonstrated that NCX in these cells is probably NACA3. The function of NCX1 in the cochlea remains obscure. A strial marginal cell is a secretory epithelial cell. In this cell, NACA3 is possibly associated with calcium transport. In hair cells NACA3 might be responsible for extruding the calcium that enters the cells during excitation. The cellular localization of multiple isoforms of NCX1 and NCX3 in the modiolus is unknown. NCX1 and NCX3 are expressed in brain (8,9,21). A spiral ganglion cell, which is a unique neuronal cell in the modiolus, probably expresses multiple isoforms of NCX1 and NCX3. In neurons, the somal calcium concentration is controlled primarily by the ATP-driven Ca2/ pump (26), but NCX1 at the nerve terminal is suggested to regulate neurotransmitter release by maintaining the intracellular calcium concentration (2). NCX might function in the nerve terminal of the spiral ganglion.
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exclusive and cassette exons underlie alternatively spliced isoforms of the Na/Ca exchanger. J. Biol. Chem. 269, 5145–5149. Ikeda, K., Saito, Y., Nishiyama, A., and Takasaka, T. (1992) Na/Ca2/ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy. Pflu¨gers Arch. 420, 493–499. Krapf, R., and Solioz, M. (1991) Na/H antiporter mRNA expression in single nephron segments of rat kidney cortex. J. Clin. Invest. 88, 783–788. Hidaka, H., Oshima, T., Ikeda, K., Furukawa, M., and Takasaka, T. (1996) The Na-K-Cl cotransporters in the rat cochlea: RT-PCR and partial sequence analysis. Biochem. Biophys. Res. Commun. 220, 425–430. Drescher, D. G., Green, G. E., Khan, K. M., Hajela, K., Beisel, K. W., Morley, B. J., and Gupta, A. K. (1993) Analysis of gaminobutyric acid A receptor subunits in the mouse cochlea by means of the polymerase chain reaction. J. Neurochem. 61, 1167–1170. Housley, G. D., Batcher, S., Kraft, M., and Ryan, A. F. (1994) Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction. Hearing Res. 75, 47–53. Housley, G. D., Greenwood, D., Bennett, T., and Ryan, A. F. (1995) Identification of a short form of the P2xR1-purinoceptor subunit produced by alternative splicing in the pituitary and cochlea. Biochem. Biophys. Res. Commun. 212, 501–508. Tachibana, M., Wilcox, E., Yokotani, N., Schneider, M., and Fex, J. (1992) Selective amplification and partial sequencing of cDNAs encoding G protein a subunits from cochlear tissues. Hearing Res. 62, 82–88. Oshima, T., Ikeda, K., Furukawa, M., and Takasaka, T. Expression of voltage-dependent chloride channels in the rat cochlea. (1997) Hearing Res. 103, 63–68. Low, W., Kasir, J., and Rahamimoff, H. (1993) Cloning of the rat heart Na/-Ca2/ exchanger and its functional expression in HeLa cells. FEBS Lett. 316, 63–67. Tsuruya, Y., Bersohn, M. M., Li, Z., Nicoll, D. A., and Philipson, K. D. (1994) Molecular cloning and functional expression of the guinea pig cardiac Na/-Ca2/ exchanger. Biochim. Biophys. Acta 1196, 97–99. Komuro, I., Wenninger, K. E., Philipson, K. D., and Izumo, S. (1992) Molecular cloning and characterization of the human cardiac Na//Ca2/ exchanger cDNA. Proc. Natl. Acad. Sci. USA 89, 4769–4773. Lee, S.-L., Yu, A. S. L., and Lytton, J. (1994) Tissue-specific expression of Na/-Ca2/ exchanger isoforms. J. Biol. Chem. 269, 14849–14852. Kofuji, P., Lederer, W. J., and Schulze, D. H. (1993) Na/Ca exchanger isoforms expressed in kidney. Am. J. Physiol. 265, F598–603. Lee, S.-L., Yu, A. S. L., and Lytton, J. (1994) Tissue-specific expression of Na/-Ca2/ exchanger isoforms. J. Biol. Chem. 269, 14849–14852. Furman, I., Cook, O., Kasir, J., and Rahamimoff, H. (1993) Cloning of two isoforms of the rat brain Na/-Ca2/ exchanger gene and their functional expression in HeLa cells. FEBS Lett. 319, 105–109. Mancini, P. M., and Santi, P. A. (1996) Immunohistochemical localization of the cardiac sodium-calcium exchange protein in the inner ear. Ann. NY Acad. Sci. 779, 400–403. DiPolo, R., and Beauge´, L. (1979) Physiological role of ATPdriven calcium pump in squid axons. Nature 278, 271–273.
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RC976533
Alternatively Spliced Isoforms of the Na//Ca2/ Exchanger in the Guinea Pig Cochlea Takeshi Oshima,1 Katsuhisa Ikeda, Masayuki Furukawa, and Tomonori Takasaka Department of Otolaryngology, Tohoku University School of Medicine 1-1, Seiryo-machi, Aoba-ku, Sendai 980-77, Japan
Received March 17, 1997
The cochlea has been suggested to express some Na// Ca2/ exchangers (NCX), since efficient acoustic transduction requires cytosolic calcium homeostasis. The present study revealed that several spliced isoforms of NCX are expressed in the guinea pig cochlea. Moreover, to determine their localization in the cochlea, microdissected RT-PCR was performed. The guinea pig cochlea was microdissected into three parts (lateral wall, the organ of Corti and modiolus). The cochlear lateral wall and the organ of Corti expressed only a single isoform of NCX1. On the other hand, five isoforms of NCX1 and four isoforms of NCX3 were detected in the cochlear modiolus. The alternative splicing may provide diverse functions for NCX in the cochlea. q 1997 Academic Press
The sodium-calcium exchanger (NCX) is an electrogenic transporter protein expressed broadly in animal cells. It is well characterized in excitable tissues, such as the heart, and mediates the exchange of one intracellular calcium ion for three extracellular sodium ions. In cardiac muscle, NCX is responsible for removing almost all of the calcium that enters via the calcium current (1). The NCX has also been shown to be present in particularly high concentrations in the neuronal synapses (2) and its mRNA is detected in the brain tissues (3) where it may play a critical role in the modulation of neuronal transmission (4). Besides these electrically excitable cells, kidney possesses the NCX which plays an important role in the regulation of Ca2/ reabsorption (5). The functional diversity of NCX in these tissues probably results from the tissue-specific expression of NCX. Up to the present, three isoforms, NCX1, NCX2 and NCX3, which are transcribed from three distinct genes, have been identified (6,7,8). Several tissue-specific isoforms of the NCX1 are known to result from alternative splicing in the long putative intracellular loop region (9). Distinct isoforms are expressed in 1
Correspondence to Takeshi Oshima. Fax: 81-22-717-7307.
heart, kidney and brain (9). NCX2 is detected only in brain and skeletal muscle (7). NCX3 is expressed in brain and skeletal muscle (8). The cochlea, which is the sound-perceptive organ, contains three functionally distinct tissues (the organ of Corti, the spiral ganglion and the stria vascularis). The organ of Corti possesses two types of hair cells both of which are auditory sensory receptors. The stria vascularis is the non-sensory ion-transporting epithelial tissue the characteristics of which are similar to those of kidney. The spiral ganglion cells are neuronal cells that transduct the sound stimulus to the brain. It is possible that NCX isoforms are expressed heterogeneously, since the cochlea is a complicated organ. Although we have previously suggested the presence of a Na//Ca2/ exchanger on the plasma membrane of a cochlear outer hair cell by electrophysiological study (10), it has not yet been determined which isoforms are expressed. The aim of the present study is to verify the presence of the NCX molecules in the guinea pig cochlea by RTPCR techniques. Moreover, their localization in the cochlea was investigated by RT-PCR, since it has been demonstrated that alternative splicing results in the presence of several tissue-specific isoforms of the NCX1 in the cardiac muscle, kidney and brain (9). The alternatively spliced exons are too short in size (the shortest is comprised of 15 nucleotides) to enable us to investigate the localization of the isoforms by immunohistochemistry and ISH. Rather, we applied RT-PCR as successfully performed in the kidney (11) to the microdissected cochlear segments, since RT-PCR for cochlear tissues has been established (12-17). We could reveal the distribution of the spliced isoforms of NCX in the three parts of the guinea pig cochlea. EXPERIMENTAL PROCEDURES cDNA synthesis and polymerase chain reaction from the whole cochlea. Young adult guinea pigs were anesthetized with diethylether and then sacrificed by decapitation. Their temporal bones were removed and the bony capsules were picked away in autoclaved phos-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS shown in Fig. 3B, were used on another nested amplification. The conditions of PCR were the same as mentioned above. To confirm the cochlear microdissection morphologically, the specimens were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The organ of Corti was mounted on a glassslide and then overlaid with a glass coverslip, followed by examination under a light microscope. The cochlear lateral wall and decalcified modiolus were postfixed with 1% osmium tetraoxide, dehydrated, and embedded in Epon 812. The samples were examined under a light microscope after thin sectioning and staining with toluidine blue.
FIG. 1. Primers. Four degenerate primers (f1, f2, r1 and r2) were synthesized based on the sequences of rat NCX1 and human NCX2. The guinea pig NCX1-specific primers (f3 and r3) were synthesized based on the sequences obtained in the present study and the guinea pig cardiac NCX. The forward and reverse primers (f4 and r4) were derived from the sequence obtained in this study (Fig.3B). Bases in parentheses indicate degeneracy.
phate-buffered saline under a stereomicroscope. Polyadenylated RNA was isolated from the whole cochlea using a mRNA isolation kit (Quick Prep Micro mRNA Purification Kit; Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions. The firststrand cDNA was synthesized from the mRNA with a commercially available kit (First-Strand cDNA Synthesis Kit; Pharmacia), in which a random hexanucleotide-primer and Molony murine leukemia virus reverse transcriptase were used. Fifteen ml of first-strand cDNA solution were obtained from two cochleas. One ml of the aliquot was used as a template for PCR. To amplify cDNA molecules of the guinea pig NCX members by RT-PCR, four degenerate primers (Fig. 1) were synthesized based on the sequences of canine (6), rat (18), and rabbit (9) NCX1 and human NCX2 (7). The forward primers from and f2 recognize the nucleotides encoding amino acids 478-486 and 530-537, and the reverse primers r1 and r2 (antisense) the amino acids 724-731 and 710-717 in the canine NCX1. Amino acid sequences in these regions are completely identical among the exchangers of the four species. The first-strand cDNA from the guinea pig cochlea and the sense and antisense primers f1 and r1 were used for RT-PCR. Thereafter, the resulting cDNA was amplified by the nested PCR with the internal primers f2 and r2. PCR was performed with a programmable thermal cycler (Model 480; Perkin Elmer, Norwalk, CT) and a thermostable Taq DNA polymerase (TaKaRa Taq; Takara Shuzo, Otsu, Japan). The initial PCR was repeated for 30 cycles as follows: 947C for 60 s, 557C for 120 s, and 727C for 120 s. The second PCR was performed under the following conditions: 947C for 45 s, 587C for 45 s, and 727C for 60 s, for 40 cycles. The procedure was completed by continuation of the last 727C incubation for a further 7 min. PCR products were analyzed by electrophoresis on 2-3 % agarose (SepRateTM-SDF, Amersham, Buckinghamshire, UK) gels.
RESULTS Whole cochlea study. PCR amplification using the guinea pig cochlear cDNA as a template was performed. The 40-cycle amplification with the primers f1 and r1 produced a faint band on an agarose gel (data not shown). To facilitate cloning the PCR product, we decided to run nested PCR with the primers f2 and r2 and the primary PCR product as a template. Agarose gel electrophoresis of the nested PCR product revealed a clear, broad band of 400-500 bp (Fig. 2). The broadness of the band suggests the heterogeneity of the PCR product. This band was not detected when the guinea pig genomic DNA was PCR-amplified under the same conditions (data not shown), indicating that this product was not derived from the genomic DNA. We performed sequencing of the fragments to confirm the heterogeneity. The PCR products were ligated to pCR II and then transformed. Hundreds of colonies were obtained. Thirty of them (gpncx1-30) were picked for plasmid isolation and restriction analysis, which revealed that 23 clones contained inserts. They were sequenced and all of them comprised open reading frames. Amino acid sequences of 7 clones were highly homologous to various spliced variants of the rabbit NCX1 (Fig.3A). The amino acid sequences of the other 16 clones were not homologous to NCX1. The sequence of the cDNA clones, gpncx8 and 12, was highly homologous to the rat NCX3 sequence (Fig.3B). The amino acid identity in this region was 97.0%. Moreover, we found three
Sequencing. The PCR products were TA-cloned into pCRTMII vector (InVitrogen, San Diego, CA). Purified plasmid DNA was sequenced bidirectionally using a fluorescent sequencing system (ALFexpress, Pharmacia). Cochlear microdissection and NCX cDNA amplification. After removing the bony cochlear wall, the lateral wall of the cochlea was peeled and collected by using fine forceps under a stereomicroscope. Next, the organ of Corti was stripped and put into a tube by the forceps. The cochlear modiolus, in which the lateral wall and the organ of Corti were excluded, was collected into a different tube. For RT-PCR, first strand cDNA was synthesized as described above. After the initial PCR with the primers f1 and r1, the nested PCR was performed with the NCX1-specific internal primers f3 and r3 (Fig. 1), based on the sequences obtained in the present study using the whole cochlea and the guinea pig cardiac NCX (19). Simultaneously, primers f4 and r4, which were derived from the sequence
FIG. 2. RT-PCR amplification of NCX in guinea pig cochlea. The nested PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide (right lane). Marker: fX174 digested with HaeIII (left lane).
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Morphological findings of cochlear microdissection. Guinea pig cochleas could be divided into three parts: 1) the cochlear lateral wall, 2) the organ of Corti and 3) the modiolus. These microdissected specimens were examined under a light microscope, which could disclose no cross-contamination. The stria vascularis, spiral ligament and spiral prominence were included in the specimen of the lateral wall (Fig.4A). The dissected Corti’s organ was observed in the Nomarski image, in which outer hair cells (OHCs) and lipid drops in Hensen’s cells could be clearly seen (Fig.4B). Deiters’ cells and pillar cells were also probably included in the specimen of the organ of Corti. In the specimen of the modiolus, the main cell-contents were spiral ganglion cells (Fig.4C). Vessels in the modiolus were observed. Few cells of the organ of Corti could be seen. Although a large amount of efferent nerve fibers were present, these fibers do not potentially contribute to RT-PCR due to the absence of ribosomes in axons.
FIG. 3. Sequencing analysis of NCX in the guinea pig cochlea. Twenty-three clones from the whole cochlea were sequenced. Polypeptide sequences are given in single-letter codes. A: Four distinct sequences were obtained from 7 clones (gpncx2, 4, 9, 16, 18, 23 and 24). Their sequences were highly homologous to rat and rabbit NCX1 sequences. Amino acids that differ from the published rat sequence are indicated by underlines. As in rabbit and rat NCX1, they were constructed by constitutive upstream and downstream sequences and alternatively spliced exons A, B, C, D and E. Their exons were named after genomic DNA sequences of rabbit NCX1 (9). The isoform NACA6 was described in rabbit brain (9). The NACA8, NACA9 and NACA10 have been identified in this study. Dashes represent gaps that were introduced in the sequences to optimize alignment. Vertical lines denote boundaries of exons. B: The amino acid sequence was obtained from 2 clones (gpncx8 and gpncx12). This was highly homologous to rat NCX3. Identities are indicated by asterisks. In 2 clones (gpncx6 and gpncx15), the underlined sequence was replaced as a possible alternative spliced exon by the sequence as follows: VHIKVIDDEAYEKNKNYFIEMMGPRMVDMSFQK. Sequencing of the other 11 clones revealed that 6 amino acids indicated by a double underline were deleted. In a clone (gpncx17), the amino acids double underlined was deleted from the sequence of gpncx6 and gpncx15. The primers f3, r3, f4 and r4 recognize the nucleotides encoding amino acids enclosed by boxes.
other cDNA isoforms due to deletion or replacement. Sequencing 11 clones revealed that 6 amino acids (ALLLSP) were deleted from the sequence shown in Fig.3B. In the other 3 clones (gpncx6, 15 and 17), the underlined sequence in Fig.3B was replaced by the sequence: VHIKVIDDEAYEKNKNYFIEMMGPRMVDMSFQK. Moreover, the 6 amino acids (ALLLSP) deletion took place in the clone gpncx17.
FIG. 4. Microdissection of guinea pig cochlea. Panel A: Stria vascularis (arrow 1), spiral ligament (arrow 2) and spiral prominence (arrow 3) are observed in the segment of the cochlear lateral wall. The lateral part of the basilar membrane (arrow 4) is seen but the organ of Corti is excluded. Panel B: On the Nomarski view, Hensen’s cells (arrow 5) and outer hair cells (arrow 6) can be easily identified in the segment of the organ of Corti. Panel C: The cochlear modiolus, in which the lateral wall and the organ of Corti were excluded, mainly contains spiral ganglion cells (arrows 7) and nerve fibers (arrow 8). BarÅ100 mm.
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FIG. 5. PCR products generated from three microdissected regions of guinea pig cochlea. Lanes 1–3: nested PCR with the primers f3 and r3. A single band of approximately 400 bp was detected in the samples of both the cochlear lateral wall (lane 1) and the organ of Corti (lane 2). On the other hand, the heterogeneous fragments were examined at a slightly higher position in the cochlear modiolus sample (lane 3). Lanes 4–6: the primers f4 and r4 were used for the nested amplification. The principal product amplified from modiolus cDNA is observed as two bands approximately 390 bp long (lane 6). This heterogeneity might be derived from alternative splicing. In lane 4 (lateral wall) or lane 5 (the organ of Corti), no band of the expected size is seen. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Markers were fX174 digested with HincII (lanes M ).
Localization of NCX1 isoforms in cochlea. To examine the localization of NCX1 molecules among the three cochlear parts, the nested PCR was performed with the nested primers f3 and r3. The PCR products generated from the three microdissected regions were first checked by agarose gel electrophoresis (Fig. 5). The amplified products from the lateral wall and the organ of Corti were detected as a single band. A relatively broad band at a position higher than those from the lateral wall and the organ of Corti was observed in the sample of the modiolus. Sequencing analysis was next performed. In all the clones from the lateral wall (nÅ8) and the organ of Corti (nÅ5) the exons B and D were expressed, whereas the other exons A, C and E, were excluded. Five isoforms, of which the exon patterns were ACDE (nÅ4), AD (nÅ2), ADE (nÅ1), BD (nÅ2) and BDE (nÅ3), were found by sequencing 12 clones from the modiolus. Localization of putative NCX3 in cochlea. A sequence highly homologous to rat NCX3 was detected in the guinea pig cochlea by RT-PCR in this study. The cochlear localization of this molecule was investigated by RT-PCR with the microdissected cochlear tissues. The nested PCR with the primers f4 and r4 produced a band of the expected size from the modiolar tissue, but not from the cochlear lateral wall or the organ of Corti. DISCUSSION This study revealed that multiple isoforms of NCX were expressed in the guinea pig cochlea. It is considered that NCX protein has 11 putative transmembrane domains and a long intracellular loop domain between
the 5th and 6th transmembrane domains (20). In this intracellular loop, alternative splicing takes place. The rabbit (9) and rat (21) NCX1 has six alternatively spliced exons designated as A-F in this region. Our sequencing analysis of this region demonstrated that the exons of the guinea pig NCX1 are almost identical to those of the rabbit and rat NCX1. We found five exons corresponding to exons A-E, however, exon F could not be detected in this study. This result does not indicate a lack of exon F in the guinea pig NCX1, since the guinea pig cardiac NCX1 possesses exon F (19). Rather, alternative splicing might possibly exclude exon F in the guinea pig cochlea. In this study, the expressed exon patterns of seven whole cochlear clones were ACDE (nÅ3), ADE (nÅ2), ACD (nÅ1) and BDE (nÅ1). The pattern ACD (the clone gpncx9) has been described as NACA6, which is expressed in the brain (9). Other patterns, ACDE, ADE and BDE, which have never appeared in other tissues or species, corresponded to novel spliced variants. We designated them as NACA8, NACA9, and NACA10, respectively. We could detect the clones gpncx8 and gpncx12 which were not highly homologous to NCX1. Rather, they were similar to rat NCX3. Since the amino acid identity in this region was 97.0%, this sequence is presumed to be guinea pig NCX3. We report here 14 other clones which are considered to be spliced variants of gpncx8 and gpncx12. No sequences homologous to human NCX2 were found in this study. The RT-PCR following the cochlear microdissection revealed the cochlear localization of spliced isoforms of NCX1 and NCX3. NCX3 messages could be detected only in the modiolus. Spiral ganglion cells in the modiolus possibly express NCX3 since high levels of it are expressed in brain (8). With respect to NCX1, the expressed exon pattern BD was detected in all three cochlear parts. This isoform is described as NACA3, which is expressed predominantly in kidney (22). It is additionally expressed in eye, aorta, intestine and thymus (23). The expressed exon patterns in the modiolus were ACDE (NACA8), AD, ADE (NACA9) and BDE (NACA10), besides BD (NACA3). The isoform AD, which was first isolated from rat brain (24), has been described as NACA4 (9). NACA6 obtained from the whole cochlea (gpncx9) could not be detected by the microdissected cochlear RT-PCR. This might be rarely expressed in cochlea. PCR products obtained from the whole cochlea seemed to be derived from the modiolus, not from the lateral wall or the organ of Corti. This result may be explained by the fact that the number of cells in the modiolus is greater than those in the lateral wall and the organ of Corti. Whole cochlear RTPCR is unlikely to reflect messages derived from specific cochlear regions such as the stria vascularis and the organ of Corti. Therefore, microdissected rather than whole tissue RT-PCR in the cochlea is useful to investigate the localization of messages.
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We did not demonstrate the cellular distribution of NCX in the cochlea, however, immunohistochemical and physiological investigations have given us some information on the cochlear localization of NCX. Na// Ca2/ exchange activity has been detected in cochlear outer hair cells using fluorescent probes (10). A monoclonal antibody against NCX1 has recognized strial marginal cells and Hensen’s cells (25). Our RT-PCR demonstrated that NCX in these cells is probably NACA3. The function of NCX1 in the cochlea remains obscure. A strial marginal cell is a secretory epithelial cell. In this cell, NACA3 is possibly associated with calcium transport. In hair cells NACA3 might be responsible for extruding the calcium that enters the cells during excitation. The cellular localization of multiple isoforms of NCX1 and NCX3 in the modiolus is unknown. NCX1 and NCX3 are expressed in brain (8,9,21). A spiral ganglion cell, which is a unique neuronal cell in the modiolus, probably expresses multiple isoforms of NCX1 and NCX3. In neurons, the somal calcium concentration is controlled primarily by the ATP-driven Ca2/ pump (26), but NCX1 at the nerve terminal is suggested to regulate neurotransmitter release by maintaining the intracellular calcium concentration (2). NCX might function in the nerve terminal of the spiral ganglion.
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