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The inner ear is essential for maintaining balance and hearing predator and prey in the environment. Each inner ear contains three CaCO3 otolith polycrystals, which are calcified within an alkaline, K+-rich endolymph secreted by the surrounding epithelium. However, the underlying cellular mechanisms are poorly understood, especially in marine fish. Here, we investigated the presence and cellular localization of several ion-transporting proteins within the saccular epithelium of the Pacific Chub Mackerel (Scomber japonicus). Western blotting revealed the presence of Na+/K+-ATPase (NKA), carbonic anhydrase (CA), Na+-K+-2Cl--co-transporter (NKCC), vacuolar-type H+-ATPase (VHA), plasma membrane Ca2+ ATPase (PMCA), and soluble adenylyl cyclase (sAC). Immunohistochemistry analysis identified two distinct ionocytes types in the saccular epithelium: Type-I ionocytes were mitochondrion-rich and abundantly expressed NKA and NKCC in their basolateral membrane, indicating a role in secreting K+ into the endolymph. On the other hand, Type-II ionocytes were enriched in cytoplasmic CA and VHA, suggesting they help transport HCO3- into the endolymph and remove H+. In addition, both types of ionocytes expressed cytoplasmic PMCA, which is likely involved in Ca2+ transport and homeostasis, as well as sAC, an evolutionary conserved acid-base sensing enzyme that regulates epithelial ion transport. Furthermore, CA, VHA, and sAC were also expressed within the capillaries that supply blood to the meshwork area, suggesting additional mechanisms that contribute to otolith calcification. This information improves our knowledge about the cellular mechanisms responsible for endolymph ion regulation and otolith formation, and can help understand responses to environmental stressors such as ocean acidification.
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Molecular and biochemical characterization of the bicarbonate sensing soluble adenylyl cyclase from a bony fish, the rainbow trout Oncorhynchus mykiss
Soluble adenylyl cyclase (sAC) is a HCO3 -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid–base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by HCO3 at physiologically relevant levels for fish internal fluids (EC50∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid–base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.
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- Award ID(s):
- 1754994
- PAR ID:
- 10222783
- Date Published:
- Journal Name:
- Interface focus
- Volume:
- 11
- Issue:
- 2
- ISSN:
- 2042-8901
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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