The heat shock protein response and physiological stress in aquatic organisms
Creators: name: Mazur, Carl François
Publisher: name: University of British Columbia
Year: 2009

description: The experiments described in this thesis were performed to examine the relationship between heat-shock proteins (hsp) and the hormonal stress response in aquatic animals. Cell lines were first exposed to selected stressors to determine which heat shock proteins are induced by environmental stressors. The hsp70 and hsp30 responses were then measured in the tissues of live fish pre-exposed to a 45s handling stress, intraperitoneal Cortisol implants or heat shock to test the effect physiological stress and Cortisol on the heat shock protein response. Heat shock protein levels were quantified by densitometric scanning of Western blots probed with salmonid-specific rabbit polyclonal antibodies. The stressors used for these experiments were 10 and 15°C heat shocks, pH 4.5 to pH 10, 10 mM L-azetidine, and 45 s physical handling. Autoradiographs of one and two dimensional SDS-PAGE gels showed, with few exceptions, that new 70 and 30 kDa proteins were synthesized by cell lines exposed to the selected stressors. A further investigation of radiolabeled proteins in Chinook salmon embryonic (CHSE) cell line exposed toa wide pH range, from pH 4.5 to pH 10, showed a prominent 70 kDa protein in response to either low or high pH. A novel 80 kDa protein was observed 1 h and 3 h after exposure of the cells to pH 10.0, and a 32 kDa protein band was observed in cells exposed to pH 5.5. The physiological stress response was induced in live fish by physical handling. Circulating plasma Cortisol levels were increased alone by intraperitoneal slow-releasing Cortisol implants. Handling and Cortisol implants resulted in elevated plasma Cortisol and glucose levels. Plasma sodium, chloride and potassium ion levels were essentially unaffected by any of the treatments. Gill, anterior kidney, liver and skeletal muscle samples were taken at regular intervals heat shock treatments and showed elevated hsp70 levels in the gill, liver, and kidney tissues 1 h after a 2-h 10°C step change heat shock. The four tissues, including skeletal muscle, had elevated hsp70 concentrations 48 h after heat shock. Only the gill tissues had elevated hsp30 levels 1 h after heat shock and no tissues had elevated hsp30 levels 48 h after the heat shock. Hsp70 levels were elevated in gills tissues for three weeks after the fish were exposed to a 2-h 15°C step change heat shock and returned to ambient temperature. Physical handling did not induce increased levels of hsp70 in gill, liver or kidney tissues. Handling the fish prior to heat shock suppressed the typical hsp70 level increase observed in the gills of heat-shocked fish, however, elevated plasma Cortisol concentrations alone did not attenuate the gill hsp70 increase caused by heat shock. Gill tissues from fish which were handled or cortisol-injected prior to heat shock showed no increase in hsp30 concentration either 1 or 48 h after the heat shock. Primary cultures of trout hepatocytes were exposed to physiological (10⁻⁷) and pharmacological (10⁻⁵) concentrations of adrenaline to test for its effect on heat shock protein levels in vitro. Propranolol was used to test the effect of blocking U-receptors during exposure of hepatocytes to adrenaline. These hepatocyte exposures showed that cells exposed to physiological or pharmacological concentrations of adrenaline had elevated levels of hsp70. Cells exposed to equimolar pharmacological (10⁻⁵) concentrations of both adrenaline and the p-blocker propranolol had the same hsp70 concentrations as those found in control cells. The blocking effect of propranolol on the ϐ-receptors may therefore block the intracellular alkalosis known to be caused by adrenaline, and which may have induced the hsp70 in adrenaline exposed primary hepatocytes.