Isolation and characterization of a larval crustacean salt gland from the brine shrimp Artemia salina Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/00000348s

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  • A method for isolating the salt gland (SG) from the larval brine shrimp has been developed. This extra-renal organ is removed from 5-10% of Stage Inauplii by vortexing after incubating the animals at 37°C for 8 hours in a saline solution buffered with 100 mM sodium phosphate at pH 7.6. Removal is dependent on the use of a specific limited set of incubation conditions which have been defined and optimized in this study. Subsequent to removal, the SG are separated from extraneous material by passing the preparation through a series of fine nylon mesh screens and a glass bead column. This protocol consistently produces approximately 5000 isolated SG, representing 250,000 secretory cells, which are 70-100% of the material present. Light, scanning, and transmission electron microscopic examination demonstrated that the organ is removed intact with good preservation of both cellular and subcellular structure. Approximately two-thirds of the twenty-eight morphological features considered, showed little or no change from in vivo. Alterations included enlargement of the tubular labyrinth lumens, loss of some definition by mitochondria cristae and alteration of cell contacts including disruption of apical cell junctions. Cell integrity of the isolated salt gland cells was confirmed by functional studies. The vital dyes, eosin, trypan blue, and nigrosin were excluded by 95% of the cells, stored at either 22°C or 4°C, for at least 12 hours. The oxygen consumption rate was 22.7 mM 0₂ (min mg protein)⁻¹, and could be altered predictably by compounds known to affect oxidative phosphorylation and ion transport. The specific activity of Na+K ATPase was 9.1 mM P (hr mg protein)⁻¹, which is 17% of the total body enzyme activity. Experiments to measure ³H-ouabain binding were unsuccessful. Preliminary experiments to localize the Na+K ATPase using potassium-nitrophenylphosphatase cytochemical methods showed unusual results, including reaction products on both basolateral and apical membranes and outer mitochondria membranes, which require further investigation. These studies demonstrate that the most important structural and functional characteristics of the SG are retained and therefore the isolated SG preparation is useful for in vitro study of this unique crustacean ion secreting epithelium.
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