The effects of extracellular sodium chloride on the activity and expression of Na,K-ATPase in primary cultures of dogfish (Scyliorhinus canicula) rectal gland epithelial cells
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Dogfish, Scyliorhinus canicula, rectal gland epithelial cells were successfully cultured using two different techniques: 1) a perfusion based technique and 2) a modified Valentich's technique. The morphology of the primary rectal gland epithelial cell cultures was investigated using light, fluorescence and electron microscopy. These studies demonstrated that the cell cultures express most of the structural features of native shark rectal gland cells, including numerous mitochondria, complex tight junctions and extensive membrane folding. The cultured cells using the perfusion technique adopted an extremely flattened morphology when grown on collagen. These cells whether grown in suspension or on collagen, displayed a striking level of vacuole formation, these vacuoles were not associated with transport epithelia. The rectal gland cell cultures were then used to investigate the effect increasing extracellular sodium chloride concentration has on rectal gland cells Na, K-ATPase activity. Increasing sodium chloride concentration in the growth medium by 50% (240 mM to 360 mM) resulted in a transient 3-4 fold increase in Na, K-ATPase activity in cell homogenates approximately 12 hours after the medium change. The response was dependent upon both sodium and chloride ions and was also inhibited by the loop diuretic bumetanide (0.1 mM within 30 minutes), indicating that entry of the ions into the cell is via the Na, K, C1 cotransporter. Incubation of cells in normal medium in the presence of the sodium ionophore monensin also resulted in a dose dependant sustained increase in Na, K-ATPase activity following a 12 hour incubation. The increase in Na, K-ATPase activity associated with increased extracellular sodium chloride concentration was only seen in cells grown on collagen and not in cells grown in suspension. Increases in activity are sensitive to the protein synthesis inhibitor cycloheximide (10 mug/ml), but not the transcriptional inhibitor actinomycin D suggesting that up-regulation of the Na, K-ATPase occurs at the level of translational regulation. Unfortunately this result could not be confirmed using Northern analysis due to unforeseen difficulties in extracting sufficient RNA from the cell cultures. Addition of bumetanide (0.1 mM) to cells grown in normal medium caused a rapid but reversible down-regulation (by 70%) of basal Na, K-ATPase activity within 30 minutes. The anti-microtubular agent colchicine (0.1 mug/ml) inhibited the bumetanide induced down-regulation of Na, K-ATPase and also the recovery of activity following bumetanide removal. The rectal gland cell cultures were used to investigate potential hormonal regulators of the shark rectal gland. The effect of the putative regulators of sodium chloride secretion scyliorhinin II and sCNP on intracellular concentrations of cAMP and cGMP was investigated. The cell cultures were shown be hormonally active as they responded with an increase in intracelllular cAMP concentration to forskolin, PGE1 and PGE2. When scyliorhinin II (10 muM) and IBMX (1 mM) was perfused through the isolated rectal gland a 2 fold increase in cAMP concentration was found in the perfusate after 8 minutes, however no increase was seen in cAMP levels when cell cultures were treated with scyliorhinin II. Shark CNP increased cGMP concentrations in the perfusates of the perfused rectal gland by up to four fold after seven minutes but there was no consistent effect on cGMP concentrations in the cultured cell monolayer. In conclusion it is believed that sCNP and scyliorhinin II mediate their actions on the regulation of sodium chloride secretion by the rectal gland at the vascular level, controlling the extent of perfusion of the gland. This study showed that high salt levels in the medium of shark rectal gland cell monolayers increased the measurable Na, K-ATPase activity and that this response was dependent on protein synthesis but not transcription. It also showed that the response is inhibited by the loop diuretic bumetanide, indicating that entry of the ions into the cell is via the Na, K, Cl cotransporter and that the increase in Na, K-ATPase activity is presumably due to an increase in intracellular sodium concentration. The hormones sCNP and scyliorhinin II appear to mediate their actions on the regulation of sodium chloride secretion by the rectal gland at the vascular level controlling the extent of perfusion of the gland. In conclusion although sodium chloride transport in the dogfish rectal gland requires much more investigation, this study has hopefully proved that dogfish epithelial cell cultures provide a good model for further investigations involving the regulation of activity and expression of the sodium pump.
Thesis, PhD Doctor of Philosophy