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Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells.

Liao BK, Deng AN, Chen SC, Chou MY, Hwang PP - BMC Genomics (2007)

Bottom Line: Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills.The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, ROC. lbk@ecophysiology.org.tw

ABSTRACT

Background: Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data.

Results: Using a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.

Conclusion: The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.

No MeSH data available.


Double fluorescence in situ hybridizations of zecac/zpmca2 (A-C) and zecac/zncx1b (D-F) in frozen sections of zebrafish gills. A and D, zecac mRNA; B, zpmca2 mRNA; C, merged image of A and B; E, zncx1b mRNA; F, merged image of D and E. Arrow indicates the colocalization of zecac/zpmca2 (A-C) and zecac/zncx1 (D-F), respectively. An asterisk (*) indicates the zpmca2 signal without zecac colocalization (A and B) and the zncx1b signal without zecac colocalization (D and E). Scale bar = 20 μm.
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Figure 7: Double fluorescence in situ hybridizations of zecac/zpmca2 (A-C) and zecac/zncx1b (D-F) in frozen sections of zebrafish gills. A and D, zecac mRNA; B, zpmca2 mRNA; C, merged image of A and B; E, zncx1b mRNA; F, merged image of D and E. Arrow indicates the colocalization of zecac/zpmca2 (A-C) and zecac/zncx1 (D-F), respectively. An asterisk (*) indicates the zpmca2 signal without zecac colocalization (A and B) and the zncx1b signal without zecac colocalization (D and E). Scale bar = 20 μm.

Mentions: Subsequently, double-fluorescence in situ hybridizations were used for zecac and zpmca2 (Figure 7A–C), and zecac and zncx1b (Figure 7D–F), respectively. More gill cells expressed zpmca2 and zncx1b mRNAs than zecac mRNA (Figure 7A–F). Notably, zpmca2 and zncx1b were also abundantly expressed in gill lamellae, where fewer MR cells are usually found [24]. zecac and zpmca2 mRNAs were colocalized only in a portion of gill cells, but were not colocalized all of the time. Around half of the zecac-positive cells co-expressed zpmca2 signals (51.7 +/- 3.50%, n = 3), while only 40.3 +/- 1.35% (n = 3) of zpmca2-positive cells showed zecac signals (Figures 7A–C, 8). Similar results were found in the case of double labeling of zecac and zncx1b (Figures 7D–F, 8). The proportion of zncx1b-positive cells that co-expressed zecac was 77.1 +/- 1.35% (n = 3), while zncx1b signals could be detected in all zecac-positive cells (Figures 7D–F, 8).


Expression and water calcium dependence of calcium transporter isoforms in zebrafish gill mitochondrion-rich cells.

Liao BK, Deng AN, Chen SC, Chou MY, Hwang PP - BMC Genomics (2007)

Double fluorescence in situ hybridizations of zecac/zpmca2 (A-C) and zecac/zncx1b (D-F) in frozen sections of zebrafish gills. A and D, zecac mRNA; B, zpmca2 mRNA; C, merged image of A and B; E, zncx1b mRNA; F, merged image of D and E. Arrow indicates the colocalization of zecac/zpmca2 (A-C) and zecac/zncx1 (D-F), respectively. An asterisk (*) indicates the zpmca2 signal without zecac colocalization (A and B) and the zncx1b signal without zecac colocalization (D and E). Scale bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2140269&req=5

Figure 7: Double fluorescence in situ hybridizations of zecac/zpmca2 (A-C) and zecac/zncx1b (D-F) in frozen sections of zebrafish gills. A and D, zecac mRNA; B, zpmca2 mRNA; C, merged image of A and B; E, zncx1b mRNA; F, merged image of D and E. Arrow indicates the colocalization of zecac/zpmca2 (A-C) and zecac/zncx1 (D-F), respectively. An asterisk (*) indicates the zpmca2 signal without zecac colocalization (A and B) and the zncx1b signal without zecac colocalization (D and E). Scale bar = 20 μm.
Mentions: Subsequently, double-fluorescence in situ hybridizations were used for zecac and zpmca2 (Figure 7A–C), and zecac and zncx1b (Figure 7D–F), respectively. More gill cells expressed zpmca2 and zncx1b mRNAs than zecac mRNA (Figure 7A–F). Notably, zpmca2 and zncx1b were also abundantly expressed in gill lamellae, where fewer MR cells are usually found [24]. zecac and zpmca2 mRNAs were colocalized only in a portion of gill cells, but were not colocalized all of the time. Around half of the zecac-positive cells co-expressed zpmca2 signals (51.7 +/- 3.50%, n = 3), while only 40.3 +/- 1.35% (n = 3) of zpmca2-positive cells showed zecac signals (Figures 7A–C, 8). Similar results were found in the case of double labeling of zecac and zncx1b (Figures 7D–F, 8). The proportion of zncx1b-positive cells that co-expressed zecac was 77.1 +/- 1.35% (n = 3), while zncx1b signals could be detected in all zecac-positive cells (Figures 7D–F, 8).

Bottom Line: Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills.The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, ROC. lbk@ecophysiology.org.tw

ABSTRACT

Background: Freshwater fish absorb Ca2+ predominantly from ambient water, and more than 97% of Ca2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca2+ uptake in gill MR cells, Ca2+ passively enters the cytosol via the epithelium Ca2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data.

Results: Using a strategy of BLAST from the zebrafish genome database (Sanger Institute), 6 isoforms of PMCAs (PMCA1a, PMCA1b, PMCA2, PMCA3a, PMCA3b, and PMCA4) and 7 isoforms of NCXs (NCX1a, NCX1b, NCX2a, NCX2b, NCX3, NCX4a, and NCX4b) were identified. In the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, 5 PMCAs and 2 NCXs were ubiquitously expressed in various tissues including gills. Triple fluorescence in situ hybridization and immunocytochemistry showed the colocalization of zecac, zpmca2, and zncx1b mRNAs in a portion of gill MR cells (using Na+-K+-ATPase as the marker), implying a subset of ionocytes specifically responsible for the transepithelial Ca2+ uptake in zebrafish gills. The gene expressions in gills of high- or low-Ca2+-acclimated zebrafish by quantitative real-time PCR analysis showed that zecac was the only gene regulated in response to environmental Ca2+ levels, while zpmcas and zncxs remained steady.

Conclusion: The present study provides molecular evidence for the specific isoforms of Ca2+ transporters, zECaC, zPMCA2, and zNCX1b, supporting the current Ca2+ uptake model, in which ECaC may play a role as the major regulatory target for this mechanism during environmental challenge.

No MeSH data available.