Stress Responses in Fish Tissue
The aim of this project is to transfer and utilize the knowledge of a number of physiological processes from mammalian cells to fish muscle cells. The intention is to study physiological processes in fish muscle that cause or influence post mortem changes, such as rigor mortis, changes in water holding ability, production of free fatty acids and breakdown of proteins, in order to reveal its implications for fish processing and product quality.
1. PRESENT STATE OF KNOWLEDGE AND OBJECTIVES
Fish quality and ischaemia
There is great similarity between the processes post mortem in the fish muscle and the physiological processes in living organisms/cells suffering from lack of oxygen. A number of the physiological processes is a direct initiation of the post mortem processes. It will, therefore, be important to know the corresponding physiological reaction pattern at ischaemia in fish muscle cells and know how they are regulated.
Cell physiological background
Else Hoffmanns group at the August Krogh Institute has a comprehensive background knowledge on regulation of cellular water content, cellular calcium and cellular pH (e.g. Hoffmann and Dunham, 1995, Hoffmann and Pedersen, 1998 and Hoffmann and Mills, 1999). In addition the group has previously worked with the effect of anoxia in mouse neocortical neurons on calcium, pH and cellular water (Jørgensen et al. 1999).
The available knowledge of the relation between ischemic/anoxic cell death and cell volume control is important for the present project and will briefly be reviewed here. Ischaemia will usually lead to a decrease in the cellular pH, one of the reasons being a rise in acid production via glycolysis.
The acidification stimulates a transport system in the cell membrane, which exchanges protons with sodium resulting in an increase in the cellular sodium concentration. Another reason for an increase in sodium is that active extrusion of sodium from the cells via the sodium/potassium pump is inhibited from the lack of energy in the ischemic cells. Chloride ions follow the sodium uptake and the muscle thus absorbs salt and then water. This initial cell swelling deteriorates the flux of oxygen and expedites cell death. Cell swelling is also known to stimulate phospholipase A2, which then releases the polyunsaturated fatty acid arachidonic acid from the phospholipids in the membrane. The arachidonic acid can be transformed into prostaglandins and leukotrienes both of which are harmful to the muscle, or it can as a free fatty acid be more susceptible to oxidation (increased rancidity in fish). At the same time calcium in the cells will increase, which has a long series of consequences. Calcium also stimulates phospholipases, which releases more fatty acids. Furthermore, calcium induces contraction of the muscle and is therefore also involved in rigor mortis. Cell swelling and the increase of calcium in the cell will then lead to an opening of the potassium and chloride channels in the membranes, whereupon the ions leave the cell followed by water.
This means that after the initial swelling of the muscles you find a secondary considerable loss of ions and fluid from the cells. This loss of liquid from the cells start a long series of processes, known as apoptose or programmed cell death (e.g. Lang et al. 1998). During this process major changes of the cellular proteins occur. Some proteins are phosphorylated and others are dephosphorylated and in the later stages of the cell death process the proteins are broken down by proteases
2. EXPERIMENTAL DESIGN
In the project we want to examine the correlation between lack of oxygen in muscle cells, cellular acidification, increase of cellular calcium, change in cell volume and ion channel activity. In addition we want to characterize the protein kinases/phosphatases that are activated in response to the ischemic stress situation and to follow changes in the proteomic pattern of the muscle cells as a function of time after start of the ischemic stress. It is the intention to obtain a thorough understanding of the relationship between these factors. This can be obtained:
- by establishing permanent cell lines of fish myocytes.
- by adapting methods for quantification of calcium in the free cytoplasm as well as in the sarcoplasmatic reticulum to the fish muscle cells.
- by developing methods to measure pH in the fish muscle cells.
- by examining a number of parameters in fish muscle cells in culture after ischemia. These include:
- the effect on intracellular pH and Ca2+ by means of fluorescence methods and
confocal laser scanning microscope. As previously done with mammal cells
(Pedersen et al. 1997; Jørgensen et al. 1999).
- channel activation as measured by whole cell patch clamp technique.
- protein phosphorylations and protein dephosphorylations as well as protein break
down in the fish muscle evaluated by two-dimensional gel electrophoresis
(Jessen 1996) and western blotting.
The project will to a great extent require development of the methods to be used, but should be realistic seen in the light of previous experiences, where Else Hoffmann's group (AUGUST KROGH GROUP) has great experience with fluorescence methods, confocal laser scanning microscope and patch clamp technique, while Flemming Jessen (DIFRES GROUP) has established two-dimensional gel electrophoresis of fish muscle proteins.
2. 2. Methods in more detail
The cytoskeleton: To study the F-actin organization cells will be fixed, incubated with rhodamine-conjugated phalloidine and viewed using our new confocal laser scanning microscope (CLSM).
The free cytosolic Ca2+ concentration and cellular pH: pH and [Ca2+]i will be monitored simultaneously using Fura-2 and BCECF (fluorescent probes) and determined in single cells using either fluorescence microscopy and digital image processing as previously described for neuronal cells (Jørgensen et al. 1999) or confocal laser scanning microscopy (CLSM) using a UV laser.
Protein kinases and phosphatases
(i) Protein phosphorylation and dephosphorylation will be investigated by 2D gel electrophoresis identified by image analysis. (ii) The role of various protein kinases and phosphatases will be addressed using specific inhibitors of protein kinases and phosphatases.
Gluthatione and oxygen radicals
I will investigate whether an eventual increased release of arachidonic acid and leukotrienes during ischeamic cell swelling of the fish muscle cells is accompanied by an increase in superoxide anions (*O2-) and a reduction of GSH. GSH levels will be estimated by a commercial assay kit and the super anion *O2- will be detected by a luminol chemiluminescent assay.
2D-gel electrophoresis
for separation of proteins in the first dimension we will use the methods of Görg et al. 1988, based on isoelectric focusing in an immobilized pH gradient. This method gives highly reproducible protein patterns, and is the most widely used technique today. The second dimension will be performed as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 2D gels will be compared by image analysis using the software PDQUEST (BIO-RAD). Candidate phospholipases and protein kinases/phosphatases will be excised from the gels and identified if possible by mass spectrometry and protein sequencing (partly as an external service as non of the supervisors at the moment have access to relevant equipment for mass spectrometry; Flemming Jessen takes part in applications for such equipment [co-ordinated by Emøke Bendixen, Research Centre Foulum] ).
References
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Görg, A., W. Postel & S. Günther (1988) Electrophoresis 9, 531-546
Hoffmann, E.K., and Dunham, P. B. (1995). Membrane mechanisms and intracellular signalling in cell volume regulation. Int.Rev.Cytol. 161: 173-262.
Hoffmann, E.K. & S.F. Pedersen (1998). Sensors and signal transduction in the activation of cell volume regulatory ion transport systems. In: Cell volume regulation. Lang, F. (ed.). Contrib. Nephrol. 123: 50-78, Karger, Basel.
Hoffmann, E.K. & J. Mills (1999). Membrane events involved in volume regulation. Current Topics in Membranes 48: 123-196.
Huss,H.H. (1995). Quality and quantity changes in fresh fish. FAO Fisheries Technical Paper 348.
Jessen, F. (1996). Freezing induced protein changes in fish evaluated by two-dimensional gel electrophoresis. In: Refrigeration and Aquaculture. Proceedings of the meeting of commission C2. Bordeaux, March 20-22, 1996. pp. 363-370.
Jørgensen, N.K, S.F. Pedersen, I. Damgaard, A. Schousbo & E.K. Hoffmann (1999). Increases in Ca and changes in intracellular pH during chemical anoxia in mouse neocortical neurones in primary culture. J. Neuroscience Research 56: 358-370.
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Lang, F., A. Leppe-Wienhues, I. Szabo, D. Siemen & E. Gulbins (1998). Cell volume in cell proliferation and apoptotic cell death. In: Cell volume regulation. Lang, F. (ed.). Contrib. Nephrol.123: 158-168, Karger, Basel.
Pedersen, S.F., N.K. Jørgensen, I. Damgård, A. Schousboe, E.K. Hoffmann (1997). Mechanisms of phi regulation studied in individual neurones cultured from the mouse cerebral cortex. J. Neurosci.Res. 51: 431-441.
Pedersen, S.F., N.K. Jørgensen & E.K. Hoffmann (1998). Dynamic of Ca and pH in Ehrlich ascites tumour cells after Ca mobilizing agonists or exposure to hypertonic solution. Eur. J. Physiol. 436: 199-210.
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Thoroed, S.M., L. Lauritzen, I.H. Lambert, H.S. Hansen & E.K. Hoffmann (1997). Cell swelling activates phospholipase A2 in Ehrlich ascites tumour cells. J. Membrane Biol. 160: 47-58.