In an investigation aimed at clarifying the mechanism of crystal dissolution of the calcium carbonate lattice in otoconia (the mineral particles embedded in the otolithic membrane) of the endolymphatic sac (ELS) of the bullfrog, cDNAs encoding the A-and E-subunits of bullfrog vacuolar protonpumping ATPase (V-ATPase) were cloned and sequenced. The cDNA of the A-subunit consisted of an 11-bp 5'-untranslated region (UTR), a 1,854-bp open reading frame (ORF) encoding a protein comprising 617 amino acids with a calculated molecular mass of 68,168 Da, and a 248-bp 3'-UTR followed by a poly(A) tail. The cDNA of the E-subunit consisted of a 72-bp 5'-UTR, a 681-bp ORF encoding a protein of 226 amino acids with a calculated molecular mass of 26,020 Da, and a 799-bp 3'-UTR followed by a poly(A) tail. Western blot and immunofluorescence analyses using specific anti-peptide antisera against the V-ATPase A-and E-subunits revealed that these subunits were present in the ELS, urinary bladder, skin, testes, and kidneys. In the ELS, positive cells were scattered in the follicular epithelium which, as revealed by electron microscopy, corresponds to the location of mitochondria-rich cells. These findings suggest that V-ATPase, including the A-and Esubunits, exists in mitochondria-rich cells of the ELS, which might be involved in dissolution of the calcium carbonate crystals in the lumen of the ELS.

S U M M A R YIn amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog ( Rana catesbeiana ) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear. O rganisms produce hard tissues in a process called biomineralization. Hard tissue includes bones, teeth, calculus, and otoliths, which are present in many vertebrates, in scales in fish, and in eggshell in birds. In invertebrates, examples of hard tissue are the shells of mollusks and diatoms, the ossicles of sea urchins, the exoskeletons of crustaceans, and the coccoliths of coccolithophriods. Recently, several cDNAs encoding proteins involved in the formation of ear stones have been cloned and sequenced, otoconin-90 (also called otoconin-95) in mammals (Wang et al. 1998;Verpy et al. 1999;Thalmann et al. 2001) and otolith matrix protein-1 (Murayama et al. 2000) and otolin-1 (Murayama et al. 2002) in teleosts. Analysis of the molecular mechanism of the ear stone formation has advanced rapidly. In most vertebrates the endolymphatic sac, which arises as the endolymphatic duct from the junction of the utriculus and the sacculus, terminates in a small blind-ending vesicle within the braincase, whereas the amphibian endolymphatic sac not only enlarges to form processes around the brain but also extends caudally along the vertebral canal and protrudes between the vertebrae, where it is referred to as the paravertebral lime sac. The lumen of these sacs contains many tiny crystals consisting of calcium carbonate, which are formed by otoconin-22 protein. In a previous study, we found that otoconin-22 protein is present in the endolymphatic sac surrounding the pituitary gland and the paravertebral lime sac in the bullfrog (Yaoi et al. 2001). Later, we cloned and sequenced a cDNA encoding bullfrog otoconin-22 and found that calcitonin regulates the expression of otoconin-22 mRNA in the endolymphatic sac, thereby stimulating the formation of calcium crystals in the lumen of the endolymphatic sac (Yaoi et al. 2003b). Furthermore, we noted that the inner ear expressed otoconin-22 mRNA but not calcitonin receptor mRNA, although the receptor mRNA was expressed in the endolymphatic...