Molecular Cloning of Otoconin-22 Complementary Deoxyribonucleic Acid in the Bullfrog Endolymphatic Sac: Effect of Calcitonin on Otoconin-22 Messenger Ribonucleic Acid Levels

Abstract

Anuran amphibians have a special organ called the endolymphatic sac (ELS), containing many calcium carbonate crystals, which is believed to have a calcium storage function. The major protein of aragonitic otoconia, otoconin-22, which is considered to be involved in the formation of calcium carbonate crystals, has been purified from the saccule of the Xenopus inner ear. In this study, we cloned a cDNA encoding otoconin-22 from the cDNA library constructed for the paravertebral lime sac (PVLS) of the bullfrog, Rana catesbeiana, and sequenced it. The bullfrog otoconin-22 encoded a protein consisting of 147 amino acids, including a signal peptide of 20 amino acids. The protein had cysteine residues identical in a number and position to those conserved among the secretory phospholipase A(2) family. The mRNA of bullfrog otoconin-22 was expressed in the ELS, including the PVLS and inner ear. This study also revealed the presence of calcitonin receptor-like protein in the ELS, with the putative seven-transmembrane domains of the G protein-coupled receptors. The ultimobranchialectomy induced a prominent decrease in the otoconin-22 mRNA levels of the bullfrog PVLS. Supplementation of the ultimobranchialectomized bullfrogs with synthetic salmon calcitonin elicited a significant increase in the mRNA levels of the sac. These findings suggest that calcitonin secreted from the ultimobranchial gland, regulates expression of bullfrog otoconin-22 mRNA via calcitonin receptor-like protein on the ELS, thereby stimulating the formation of calcium carbonate crystals in the lumen of the ELS.

Cloning and Expression of Vacuolar Proton-Pumping ATPase Subunits in the Follicular Epithelium of the Bullfrog Endolymphatic Sac

“…Using a bullfrog PVLS cDNA library established in our previous study (Yaoi et al, 2003), we cloned cDNAs encoding the A-and Esubunits of V-ATPase. Partial cDNA fragments of bullfrog A-and Esubunits of V-ATPase were obtained by designing degenerate primers based on amino acid sequences from other species.…”

“…We performed polymerase chain reaction (PCR) analyses using cDNA prepared from the bullfrog PVLS cDNA library in 25-μl volumes of Ex-taq buffer containing 0.2 mM each dNTP and 50 pmol of each of primers 1 and 2 with 0.5 U of Ex-taq polymerase (Takara, Kyoto, Japan), as described by Yaoi et al (2003). The PCR amplification procedure consisted of an initial denaturation step at 95°C for 5 min followed by denaturation (94°C, 90 sec), annealing (50°C, 90 sec), and extension (72°C, 150 sec) for 30 cycles in a thermal cycler (ASTEC, Fukuoka, Japan).…”

“…Otoconin-22 is believed to be important in creating conditions that favor nucleation and, subsequently, in controlling the crystal growth of the calcium carbonate lattice in otoconia (mineral particles embedded in the otolithic membrane). A subsequent study by these researchers (Yaoi et al, 2003) resulted in the cloning and sequencing of a cDNA encoding bullfrog otoconin-22 and provided evidence that calcitonin regulates the expression of otoconin-22 mRNA in the ELS, thereby stimulating the formation of calcium crystals in the lumen of the ELS. These results led to the conclusion that the amphibian ELS functions as a reservoir for calcium, although the mechanism by which these crystals are dissolved remained unclear.…”

Mixing model systems: Using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development

“…In the frog Rana catesbeiana, the levels of Otoconin22(Oc22) mRNA, the major core matrix protein of aragonitic otoconia (Pote and Ross, 1991;Pote et al, 1993), have been shown to be regulated by the activity of calcitonin (Yaoi et al, 2003), suggesting that, in some animals, otoconia serve as a rapidly ionizable store of Ca 2+ that can be (reversibly) demineralized and dispersed. In humans, bone serves as a readily ionizable and regulatable source of Ca 2+ , but as shown above, many of the systems for the formation and fate of otoconia are well conserved throughout vertebrates.…”

Developmental Expression of Otoconin-22 in the Bullfrog Endolymphatic Sac and Inner Ear

“…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 sac.…”