| 1. |
- Basse, W. C., et al.
(författare)
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A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae
- 2015
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Ingår i: Comparative Biochemistry and Physiology A. - : Elsevier BV. - 1095-6433. ; 187, s. 184-192
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Tidskriftsartikel (refereegranskat)abstract
- The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50=6.5μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50=26.4μM) and furosemide (IC50=315.4μM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.
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| 2. |
- Holtmann, W. C., et al.
(författare)
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Maintenance of coelomic fluid pH in sea urchins exposed to elevated CO2: The role of body cavity epithelia and stereom dissolution
- 2013
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Ingår i: Marine Biology. - : Springer Science and Business Media LLC. - 0025-3162 .- 1432-1793. ; 160:10, s. 2631-2645
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Tidskriftsartikel (refereegranskat)abstract
- Experimental ocean acidification leads to a shift in resource allocation and to an increased [HCO3 -] within the perivisceral coelomic fluid (PCF) in the Baltic green sea urchin Strongylocentrotus droebachiensis. We investigated putative mechanisms of this pH compensation reaction by evaluating epithelial barrier function and the magnitude of skeleton (stereom) dissolution. In addition, we measured ossicle growth and skeletal stability. Ussing chamber measurements revealed that the intestine formed a barrier for HCO3 - and was selective for cation diffusion. In contrast, the peritoneal epithelium was leaky and only formed a barrier for macromolecules. The ossicles of 6 week high CO2-acclimatised sea urchins revealed minor carbonate dissolution, reduced growth but unchanged stability. On the other hand, spines dissolved more severely and were more fragile following acclimatisation to high CO2. Our results indicate that epithelia lining the PCF space contribute to its acid-base regulation. The intestine prevents HCO3 - diffusion and thus buffer leakage. In contrast, the leaky peritoneal epithelium allows buffer generation via carbonate dissolution from the surrounding skeletal ossicles. Long-term extracellular acid-base balance must be mediated by active processes, as sea urchins can maintain relatively high extracellular [HCO3 -]. The intestinal epithelia are good candidate tissues for this active net import of HCO3 - into the PCF. Spines appear to be more vulnerable to ocean acidification which might significantly impact resistance to predation pressure and thus influence fitness of this keystone species. © 2013 Springer-Verlag Berlin Heidelberg.
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| 3. |
- Ramesh, Kirti, et al.
(författare)
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Intracellular pH regulation in mantle epithelial cells of the Pacific oyster, Crassostrea gigas
- 2020
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Ingår i: Journal of Comparative Physiology B-Biochemical Systems and Environmental Physiology. - : Springer Science and Business Media LLC. - 0174-1578. ; 190, s. 691-700
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Tidskriftsartikel (refereegranskat)abstract
- Shell formation and repair occurs under the control of mantle epithelial cells in bivalve molluscs. However, limited information is available on the precise acid-base regulatory machinery present within these cells, which are fundamental to calcification. Here, we isolate mantle epithelial cells from the Pacific oyster,Crassostrea gigasand utilise live cell imaging in combination with the fluorescent dye, BCECF-AM to study intracellular pH (pH(i)) regulation. To elucidate the involvement of various ion transport mechanisms, modified seawater solutions (low sodium, low bicarbonate) and specific inhibitors for acid-base proteins were used. Diminished pH recovery in the absence of Na(+)and under inhibition of sodium/hydrogen exchangers (NHEs) implicate the involvement of a sodium dependent cellular proton extrusion mechanism. In addition, pH recovery was reduced under inhibition of carbonic anhydrases. These data provide the foundation for a better understanding of acid-base regulation underlying the physiology of calcification in bivalves.
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