Calcium ion has diverse beneficial roles in living organisms. Failure in Ca2+ homeostasis affects a variety of molecular and cellular processes, ultimately leading to many pathological consequences. In mammals, body Ca2+ homeostasis is maintained by the coordinated calcium (re)absorption that occurs in the small intestines, kidneys and bones, and is under tight hormonal control. In fish, two special organs, Corpuscles of Stannius (CS) glands and gills form a regulatory circuit to detect and regulate blood Ca2+ homeostasis. However, the underlying molecular mechanism in the regulation of gill Ca2+ uptake has not been fully examined. Moreover, some putative biological active substances in CS glands have not been identified. To address these research questions, a euryhaline fish, Japanese eel (Anguilla japonica) was used as an animal model for the study. Fish gill is equipped with epithelial calcium channel (ECaCl) as gatekeeper of Ca2+ entry, and membrane Ca2+-ATPase (PMCA) for Ca2+ efflux. To test if branchial ECaCl and PMCA responded to change in water Ca2+ level, we investigated the changes in fish adapted in artificial freshwater (AFW), Ca2+-deficient AFW (D-AFW) or high Ca2+-AFW (H-AFW). Our data illustrated both short-term and long-term effects on modulations of the transporters. The changes correlated with expression levels of stanniocalcin-1 (STC-1) in CS glands. This part of study supports the regulatory circuit between gills and the glands. In primary cell culture of Japanese eel gill cells, Ca2+ sensing was shown to be mediated by Ca2+ sensing receptor (CaSR) coupled to phospholipase C (PLC)-extracellular signal-regulated kinase (ERK) and PLC-inositol triphosphate (IP3)-Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) pathways. And CaSR-STC-1/cyclo-oxygenase-2 (COX-2) mediated protective pathway in gill cells that exerts a possible protective mechanism against an increase in intracellular Ca2+ levels associated with transepithelial Ca2+ transport. Apparently, the protective effects against Ca2+-mediated cytotoxicity of gill cell were mediated by STC-1 binding on gill cells that led to elevations of cytosolic cAMP. In a follow-up experiment of using Ca2+-imaging system in a model of thapsigargin (TG)-induced elevation of cytosolic Ca2+, a hypocalcemic action of STC-1 was demonstrated and was found to be mediated by cAMP and COX-2 pathway. To further determine the gene expressed in CS gland responsive to changes in water salinity, the first transcriptome database of CS glands from fish adapted in freshwater or seawater condition. A de novo assembly of RNA sequencing data generated 11747 unigenes and revealed 475 genes that were differentially expressed. Three functional clusters: (1) Ca2+-metabolism, (2) blood pressure and (3) ion-osmoregulation were revealed. Gene targets, in addition to STC-1 in related to the regulation of calcium metabolism and blood pressure, like calcitonin, atrial natriuretic peptide-converting enzyme and endothelin-converting enzyme 1 were identified. Taken together this thesis described a comprehensive study on the functional circuit between gills and CS glands to decipher the regulation and functions of transporters and hormones in calcium metabolism in fish.
|Date of Award||29 Aug 2014|
|Supervisor||Chris K C WONG (Supervisor)|
- Molecular biology