Oncorhynchus mykiss epithelial calcium channel (ECaC) is thought to regulate calcium uptake across the gill. ECaC is a member of the transient receptor potential (TRP) gene family. In mammals, ECaC is divided into two sub-families, TRPV5 and TRPV6. Rainbow trout appears to possess only a single ECaC gene similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis suggests that an ancestral form of the gene diverged from those of lower vertebrates prior to a gene duplication event that gave rise to TRPV5 and TRPV6.
In contrast to previous models of branchial calcium uptake which proposed an almost exclusive distribution of ECaC to mitochondrial rich cells (MRCs), the results of real time PCR on enriched gill cell populations as well as immunocytochemistry and in-situ hybridization analysis of enriched cells, cell cultures and whole gill sections suggest that ECaC is localized to a subset of both MRCs and pavement cells (PVCs).
Using real time PCR and immunocytochemistry, the response of ECaC expression to various treatments known to increase or decrease Ca2+ uptake was examined. A significant increase in ECaC mRNA expression was observed following 5 days exposure of rainbow trout to softwater ([Ca2+] = 20--30 nmol 1-1). A concurrent increase in protein expression was suggested by the immunocytochemistry results (particularly in cells located at the tips of the lamellae) and confirmed by western blotting (identifying a single immunoreactive band at 90 kDa). A significant increase (almost 100-fold) in mRNA expression was also observed following exposure to 48 h of hypercapnia (∼7.5 mm Hg; a treatment known to increase Ca 2+ uptake capacity). Immunocytochemical analysis of the hypercapnic gills suggested an increase in the apical ECaC proteins on PVCs as well as on a subset of MRCs. Treatment of fish with cortisol caused a significant increase in ECaC expression. In contrast to the above experiments, induced hypercalcemia (through infusion of CaCl2, treatment known to reduce Ca2+ uptake) resulted in a significant reduction in ECaC gene expression.
Taken together, the results presented in this thesis demonstrate that the levels of ECaC mRNA vary in direct relation to the Ca2+ transporting capacity of the gill (changes in transport capacity have previously been reported by other authors). These results thus provide evidence that, in part, Ca2+ uptake rates are regulated by the number of apical membrane Ca2+ channels that in turn modulate the inward flux of Ca2+ into gill epithelial cells. However, the data also suggests that a more in-depth characterization of gill cells is required since the distribution of ECaC appears to be more extensive than previously proposed.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/29604 |
| Date | January 2007 |
| Creators | Shahsavarani, Arash |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 127 p. |
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