title: A sea urchin Na(+)K(+)2Cl(-) cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae.
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=6.5 μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50=26.4 μM) and furosemide (IC50=315.4 μM) 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.
authors.affiliation: Physiologisches Institut, CAU Kiel, Kiel D-24098, Germany.
authors.forename: Wiebke C
authors.initials: WC
authors.lastname: Basse
authors.affiliation: Physiologisches Institut, CAU Kiel, Kiel D-24098, Germany.
authors.forename: Magdalena A
authors.initials: MA
authors.lastname: Gutowska
authors.affiliation: GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel D-24105, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Findeisen
authors.affiliation: GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel D-24105, Germany.
authors.forename: Meike
authors.initials: M
authors.lastname: Stumpp
authors.affiliation: Department of Biological and Environmental Sciences, The Sven Lovén Centre for Marine Science, University of Gothenburg, Kristineberg, Fiskebäckskil 45178, Sweden.
authors.forename: Sam
authors.initials: S
authors.lastname: Dupont
authors.affiliation: Department of Geobiology, Georg-August University of Göttingen, Göttingen D-37077, Germany.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Jackson
authors.affiliation: Physiologisches Institut, CAU Kiel, Kiel D-24098, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Himmerkus
authors.affiliation: GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel D-24105, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Melzner
authors.affiliation: Physiologisches Institut, CAU Kiel, Kiel D-24098, Germany. Electronic address: m.bleich@physiologie.uni-kiel.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Bleich
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic; D003593:Cytoplasm / Q000378:metabolism*; D004232:Diuretics / Q000494:pharmacology; D007814:Larva / Q000254:growth & development* / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D047269:Strongylocentrotus / Q000254:growth & development*
chemical_list: D004232:Diuretics; D028021:Sodium-Potassium-Chloride Symporters
keywords: Calcein; Calcification; Ion transport; Loop diuretics; NKCC (Na(+)K(+)2Cl(−) cotransporter); Strongylocentrotus droebachiensis