Biomineralisation of skeletal structures occurs in diverse marine invertebrate species. The process is dependent on the transport of seawater Ca²⁺ and HCO₃^- to the site of CaCO₃ precipitation. Sea urchin larvae are an emerging model organism for the study of the cellular mechanisms underlying calcification relevant substrate transport. These larvae possess two skeletal spicules made off high Mg-calcite by primary mesenchyme cells (PMCs) which are specialised for calcification. These cells form a syncytium which envelops individual spicules. Within the PMCs amorphous CaCO₃ is precipitated in vacuoles and subsequently transported to the growing tips of the spicule. To date, knowledge of the ion transport mechanisms is very limited, however, there are indications that furosemide can inhibit spicule calcification.

We tested the hypothesis that the Na⁺2Cl^-K⁺ cotransporter (NKCC) is directly involved in spicule calcification of Strongylocentrotus droebachiensis. Concentration dependent effects of azosemide, bumetanide and furosemide were determined and increments of spicule growth as well as morphometric parameters were measured. We show that all three loop diuretics inhibit calcification in a concentration dependent manner. Azosemide clearly had the strongest effect with an IC₅₀ of 8.1 mM versus 27.7 and 121.9 mM for bumetanide and furosemide, respectively. Spicule calcification in the prism larval stage was reduced by 51% during 48 hr exposure to 10 mM azosemide.

We conclude that NKCC1 is involved in spicule calcification and propose that it is involved in volume regulation of PMC vacuoles that precipitate CaCO₃. Ongoing work is testing the proposed cell model.