Yukio Fujino

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 microM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO3 deposition in the spicule.

Probable Role of Allylisothiocyanate‐Sensitive H+, K+‐ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus, as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H+, K+‐ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na+, H+ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [dimethylamine‐14C]‐aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K+‐dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate‐sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H+, K+‐ATPase, an H+ pump, probably mediates H+ release to accelerate CaCO3 deposition from Ca2+, CO2 and H2O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K+ permeability probably increases after the prism stage to activate H+ release.

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+‐ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere‐Derived Cells.

Embryos kept with omeprazole, a specific H+, K+‐ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro‐mere‐derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole‐sensitive H+, K+‐ATPase, an H+pump, seems to be indispensable for CaCO3 deposition (formation of spicule rod) in these spicule forming cells. H+, produced in overall reaction for CaCO3 formation: Ca2++ CO2+H2O°CaCO3+2H+, is probably released from the cells by this H+pump and hence, this reaction tends to go to CaCO3 production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H+, K+‐ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H+ejection in H+, K+‐ATPase reaction.

Changes in the Activities of H+, K+‐ATPase and Na+, K+‐ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation

In cultured cells derived from micromeres isolated at the 16‐cell stage of sea urchin embryos, the activity of H+, K+‐ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na+, K+‐ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H+, K+‐ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of 45Ca deposition in spicule rods in spicule‐forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or 45, deposition. In cultured cells exposed to nifedipine, which blocked 45Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H+, which is generated in the overall reaction to produce CaCO3 from Ca2+ and HCO3−, is probably released from the cells mainly in the reaction catalyzed by H+, K+‐ATPase to maintain successive production of CaCO3.

Development of Sea Urchin Embryos in Artificial Sea Water Containing Br−in Place of Cl−

In artificial sea water in which the Cl−concentration was reduced to less than 10% of that in normal sea water by its replacement with Br−, sea urchin eggs were fertilized and developed into abnormal plutei following almost the same time schedule as in natural sea water. These embryos had poorly developed spicules, short pluteus arms, somewhat jagged embryo‐walls and quasi‐normal archenterons. Similar embryos were obtained in another artificial sea water in which 90% of the Cl−concentration in normal sea water was reduced by Br−and 10% by acetate. In artificial sea water, in which either 90% of the Cl−was replaced by Br−or 10% was replaced by acetate, embryos developed into plutei with quasi‐normal spicules, pluteus arms and archenterons. These findings indicate that deficiency of Cl−results in somewhat abnormal sea urchin embryos. When cells derived from isolated micromeres, were cultured in these Cl−‐deficient artificial sea waters, containing Br−in place of more than 70% of the normal Cl−concentration in sea water, spicule formation was strongly inhibited, but pseudopodial cables were well developed. Thus, external Cl−seems to be necessary for at least normal formation of spicule rods.

Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus. The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose‐1‐phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased 45Ca2+ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca2+ level increases associated with fertilization‐induced Ca2+ influx and that this stimulates Ca2+ dependent protein kinase to phosphorylate phosphorylase b, resulting in an increased rate of the phosphorylase reaction.