Role of carbon dioxide and ion transport in the formation of sub-embryonic fluid by the blastoderm of the Japanese quail

1. The explanted blastoderm of the Japanese quail was used to explore the role of ions and carbon dioxide in determining the rate of sub-embryonic fluid (SEF) production between 54 and 72 h of incubation. 2. Amiloride, an inhibitor of Na+/H+ exchange, at concentrations of 10-3 to 10-6 M substantially decreased the rate of SEF production when added to the albumen culture medium. N-ethylmaleimide, an inhibitor of V type H+ ATPase, also decreased this rate but only to a small extent at the highest dose applied, 10-3 M. Both inhibitors had no effect on SEF production when added to the SEF. 3. The inhibitors of cellular bicarbonate and chloride exchange, 4-acetamido-4-'isothiocyano-2, 2-'disulphonic acid (SITS) and 4,4'diisothiocyanostilbene-2,2-'disulphonic acid (DIDS), had no effect upon SEF production. 4. Ouabain, an inhibitor of Na+/K+ ATPase, decreased SEF production substantially at all concentrations added to the SEF (10-3 to 10-6 M). Three sulphonamide inhibitors of carbonic anhydrase, acetazolamide, ethoxzolamide and benzolamide, decreased SEF production when added to the SEF at concentrations of 10-3 to 10-6 M. Benzolamide was by far the most potent. Neither ouabain nor the sulphonamides altered SEF production when added to the albumen culture medium. 5. Using a cobalt precipitation method, carbonic anhydrase activity was localised to the endodermal cells of the area vasculosa. The carbonic anhydrase activity was primarily associated with the lateral plasma membranes, which together with the potent inhibitory effect of benzolamide, suggests the carbonic anhydrase of these cells is the membrane-associated form, CA IV. 6. The changes in SEF composition produced by inhibitors were consistent with the production of SEF by local osmotic gradients. 7. It is concluded that a Na+/K+ ATPase is located on the basolateral membranes of the endodermal cells of the area vasculosa , and that a sodium ion/hydrogen ion exchanger is located on their apical surfaces. Protons for this exchanger would be provided by the hydration of CO2 catalysed by the membrane-associated carbonic anhydrase. Furthermore, it is proposed that the prime function of the endodermal cells of the area vasculosa is the production of SEF

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 − with Na+. Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 −/− mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR −/− and AE2a,b −/− mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes