scholarly journals Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium

2014 ◽  
Vol 306 (4) ◽  
pp. R211-R221 ◽  
Author(s):  
Raymond W. M. Kwong ◽  
Dan Auprix ◽  
Steve F. Perry
Keyword(s):  
In Situ Hybridization ◽  
Confocal Microscopy ◽  
Mrna Expression ◽  
Danio Rerio ◽  
Whole Body ◽  
Calcium Sensing Receptor ◽  
Larval Zebrafish ◽  
Calcium Sensing ◽  
Normal Water

The involvement of the calcium-sensing receptor (CaSR) in Ca2+ homeostasis was investigated in larval zebrafish, Danio rerio. The expression of CaSR mRNA was first observed at 3 h posfertilization (hpf) and increased with development until plateauing at ∼48 hpf. At 4 dpf, CaSR mRNA was increased in fish acclimated to low Ca2+ water (25 μM vs. 250 μM in normal water). Using immunohistochemistry and confocal microscopy, we demonstrated that the CaSR is expressed in the olfactory epithelium, neuromasts, ionocytes on the yolk sac epithelium, and corpuscles of Stannius. Results of double immunohistochemistry and/or in situ hybridization indicated that the CaSR is localized to a subset of mitochondrion-rich ionocytes enriched with Na+/K+-ATPase and epithelial Ca2+ channel ( ecac). Translational knockdown of the CaSR prevented 4 dpf larvae from regulating whole body Ca2+ levels when exposed to a low Ca2+ environment. Further, the increases in ecac mRNA expression and Ca2+ influx, normally associated with exposure to low-Ca2+ water, were prevented by CaSR knockdown. These findings demonstrate that larval zebrafish lacking the CaSR lose their ability to regulate Ca2+ when confronted with a low-Ca2+ environment. Results from real-time PCR suggested that the mRNA expression of the hypocalcemic hormone stanniocalcin ( stc-1) remained elevated in the CaSR morphants following acclimation to low-Ca2+ water. Overall, the results suggest that the CaSR is critical for Ca2+ homeostasis in larval zebrafish exposed to low environmental Ca2+ levels, possibly owing to its modulation of stanniocalcin mRNA expression.

Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio)

2009 ◽  
Vol 297 (4) ◽  
pp. R988-R997 ◽  
Author(s):  
S. F. Perry ◽  
B. Vulesevic ◽  
M. Grosell ◽  
M. Bayaa
Keyword(s):  
Mrna Expression ◽  
Danio Rerio ◽  
Whole Body ◽  
Mrna Levels ◽  
Adult Zebrafish ◽  
Chloride Uptake ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Normal Water

Experiments were performed to test the hypothesis that three members of the SLC26 anion transporter gene family (SLC26a3, A4, and A6; hereafter termed za3, za4, and za6) mediate branchial Cl−/HCO3− exchange in adult zebrafish ( Danio rerio). Real-time RT-PCR demonstrated that the gill expressed relatively high levels of za6 mRNA; za3 and za4 mRNA, while present, were less abundant. Also, za4 and za6 were expressed at relatively high levels in the kidney. The results of in situ hybridization or immunocytochemistry (za3 only) experiments performed on gill sections revealed that the SLC26 transporters were predominantly expressed on the filament epithelium (especially within the interlamellar regions) and to a lesser extent on the lamellar epithelium at the base of lamellae. This distribution pattern suggests that the SLC26 anion transporters are localized to mitochondrion-rich cells (ionocytes). Transferring fish to water containing low [Cl−] (0.02 mmol/l) resulted in significant increases in branchial SLC26 mRNA expression after 5–10 days of exposure relative to fish raised in normal water [Cl−] (0.4 mmol/l); transferring fish to Cl−-enriched water (2.0 mmol/l) was without effect on mRNA levels. Transferring fish to water containing elevated levels of NaHCO3 (10–12.5 mmol/l) caused marked increases in branchial SLC26 mRNA expression between 3 and 10 days of transfer that was associated with a significant 40% increase in Cl− uptake (as measured upon return to normal water after 7 days). A decrease in whole body net acid excretion (equivalent to an increase in net base excretion) in fish previously maintained in high [NaHCO3] water, concurrent with increases in Cl− uptake and SLC26 mRNA levels, suggests a role for these anion transporters in Cl− uptake and acid-base regulation owing to their Cl−/HCO3− exchange activities.

scholarly journals Comparative expression of the extracellular calcium-sensing receptor in the mouse, rat, and human kidney

2016 ◽  
Vol 310 (6) ◽  
pp. F518-F533 ◽  
Author(s):  
J. A. Z. Graca ◽  
M. Schepelmann ◽  
S. C. Brennan ◽  
J. Reens ◽  
W. Chang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Parathyroid Hormone ◽  
Significant Heterogeneity ◽  
Thick Ascending Limb ◽  
Calcium Sensing Receptor ◽  
Proximity Ligation ◽  
Calcium Sensing ◽  
Physiological Serum ◽  
Custom Made

The calcium-sensing receptor (CaSR) was cloned over 20 years ago and functionally demonstrated to regulate circulating levels of parathyroid hormone by maintaining physiological serum ionized calcium concentration ([Ca2+]). The receptor is highly expressed in the kidney; however, intrarenal and intraspecies distribution remains controversial. Recently, additional functions of the CaSR receptor in the kidney have emerged, including parathyroid hormone-independent effects. It is therefore critical to establish unequivocally the localization of the CaSR in the kidney to relate this to its proposed physiological roles. In this study, we determined CaSR expression in mouse, rat, and human kidneys using in situ hybridization, immunohistochemistry (using 8 different commercially available and custom-made antibodies), and proximity ligation assays. Negative results in mice with kidney-specific CaSR ablation confirmed the specificity of the immunohistochemistry signal. Both in situ hybridization and immunohistochemistry showed CaSR expression in the thick ascending limb, distal tubule, and collecting duct of all species, with the thick ascending limb showing the highest levels. Within the collecting ducts, there was significant heterogeneity of expression between cell types. In the proximal tubule, lower levels of immunoreactivity were detected by immunohistochemistry and proximity ligation assays. Proximity ligation assays were the only technique to demonstrate expression within glomeruli. This study demonstrated CaSR expression throughout the kidney with minimal discrepancy between species but with significant variation in the levels of expression between cell and tubule types. These findings clarify the intrarenal distribution of the CaSR and enable elucidation of the full physiological roles of the receptor within this organ.

Epidermal Keratinocyte Differentiation is Disrupted in Mice Lacking The Full Length Extracellular Calcium-Sensing Receptor

1997 ◽  
Vol 3 (S2) ◽  
pp. 185-186
Author(s):  
László G. Kömüves ◽  
Jonathan D. Harris ◽  
Chrystal Ho ◽  
Daniel D. Bikle
Keyword(s):  
In Situ Hybridization ◽  
Knockout Mice ◽  
Ion Homeostasis ◽  
Extracellular Calcium ◽  
Full Length ◽  
Keratinocyte Differentiation ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca2+ concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis suggests a role for CaR in local regulation of cellular functions. Although extracellular Ca2+ regulates the differentiation of keratinocytes, the role of CaR in the epidermis is not established. In this work using knockout mice lacking full length CaR, we sought to determine the role of CaR in epidermal differentiation.Dorsal skin of Casr−/− knockout mice lacking full length CaR, and Casr+/+ (wild type) control mice, aged 4 to 7 days after birth was fixed in 4% formaldehyde in PBS, and in 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer. The samples were embedded in paraffin (for immunohistochemistry and for in situ hybridization) or in Spurr’s or LR White resins. Digoxigenin labeled antisense and sense RNA probes for loricrin and filaggrin were used for in situ hybridization.

Expression of transforming growth factor alpha during development

1988 ◽  
Vol 66 (8) ◽  
pp. 1113-1121 ◽  
Author(s):  
V. K. M. Han ◽  
A. J. D'Ercole ◽  
D. C. Lee
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
Egf Receptors ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Factor Alpha

Transforming growth factors (TGFs) are polypeptides that are produced by transformed and tumour cells, and that can confer phenotypic properties associated with transformation on normal cells in culture. One of these growth-regulating molecules, transforming growth factor alpha (TGF-α), is a 50 amino acid polypeptide that is related to epidermal growth factor (EGF) and binds to the EGF receptor. Previous studies have shown that TGF-α is expressed during rodent embryogenesis between 7 and 14 days gestation. To investigate the cellular sites of TGF-α mRNA expression during development, we have performed Northern analyses and in situ hybridization histochemistry on the conceptus and maternal tissues at various gestational ages. Contrary to previous reports, both Northern analyses and in situ hybridization histochemistry indicate that TGF-α mRNA is predominantly expressed in the maternal decidua and not in the embryo. Decidual expression is induced following implantation, peaks at day 8, and declines through day 15 when the decidua is being resorbed. In situ hybridization revealed that expression of TGF-α mRNA is highest in the region of decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and embryo. In addition, we could not detect TGF-α mRNA expression in other maternal tissues, indicating that the induction of TGF-α transcripts in the decidua is tissue specific, and not a pleiotropic response to changes in hormonal milieu that occur during pregnancy. The developmentally regulated expression of TGF-α mRNA in the decidua, together with the presence of EGF receptors in this tissue, suggests that this peptide may stimulate mitosis and angiogenesis locally by an autocrine mechanism. Because EGF receptors are also present in the embryo and placenta, TGF-α may act on these tissues by a paracrine or endocrine mechanism.

Estrogen-related receptor γ2 controls NaCl uptake to maintain ionic homeostasis

2021 ◽  
Author(s):  
Shang-Wu Shih ◽  
Jia-Jiun Yan ◽  
Yi-Hsing Wang ◽  
Yi-Ling Tsou ◽  
Ling Chiu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Body Fluid ◽  
Whole Body ◽  
Ionic Homeostasis ◽  
Ion Regulation ◽  
Expression Levels ◽  
Morpholino Knockdown ◽  
Oryzias Melastigma ◽  
Euryhaline Teleost

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that ERRγ2 may play a crucial role in ion uptake. In situ hybridization and immunofluorescence staining showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl- transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl- cotransporter (NCC), the number of NCC ionocytes, Na+/Cl- influxes of ionocytes, and whole-body Na+/Cl- contents. In FW with low Na+ and low Cl-, the expression levels of mRNA for Na+/H+ exchanger 3 (NHE3) and NCC were both decreased in ERRγ2 morphants. Treating embryos with DY131, an agonist of ERRγ, increased the whole-body Na+/Cl- contents and ncc mRNA expression in ERRγ2 morphants. As such, medaka ERRγ2 may control Na+/Cl- uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

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