Induction of Heat Shock Protein Synthesis in Channel Catfish Hepatocytes
IAAAM 1991
Thomas G. Nemetz, DVM, PhD
Department of Genetics, University of Georgia, Athens, GA

Exposure of cells, tissues or intact organisms to a variety of harmful environmental stimuli elicits the production of a limited number of proteins. These were originally termed heat shock proteins (hsps) because heat shock was the first stimulus found to induce their synthesis. They are currently referred to as hsps or stress proteins.

Hsps are synthesized in both prokaryotic and eukaryotic cells and are considered to be nearly ubiquitous in nature. The proteins are categorized according to their molecular weights (range 20-110 kilo Daltons). Considerable genetic relatedness exists among proteins of similar size and between proteins of diverse species. Proteins of similar size and protein composition are designated as members of hsp families.

Differences do exist in the hsp responses of different organisms. The number of proteins synthesized in response to a particular stressor may vary among species, or within tissues of one organism. Similarly, different stressors elicit production of different subsets of hsps. Some hsps are solely stress-induced, while others are both stress-inducible and constitutively synthesized. However, the ubiquity and genetic relatedness of hsps, as well as their rapid and selective synthesis, have prompted a considerable number of investigations designed to answer two questions: what turns hsp synthesis on and off, and what do the proteins do?

Much of the research concerning hsps has been done using single-cell organisms and continuous cell lines derived from avian and mammalian species. In contrast little work has been done using fish as the laboratory animal. Examination of the hsp system could help to answer a number of questions pertinent both to the basic physiology of fish and to their successful cultivation. For instance, how similar are hsps of fish and other species? What stressors found in an aqua cultural environment induce hsp synthesis? Can induction be correlated with enhanced survival in adverse environmental conditions? Can hsp production be used as an indicator of stress in fish? Do different strains of fish differ in their ability to produce these proteins, and can such differences be useful in selection of genetically hardy brood stock?

A series of experiments were conducted to define the kinetics of hsp synthesis in cells of the channel catfish (Ictalurus puncatatus Rafinesgue). The purpose of these experiments was to determine whether stressors commonly encountered in catfish production would induce hsp synthesis, the conditions in which hsp production was optimized, and the kinetics of the hsp response. Primary cultures of hepatic cells were obtained by in situ collagenase perfusion. Cells were counted, assayed for viability and maintained in replicate cultures for 24 hours prior to stressor stimulus. Stressors which were evaluated included heat, recovery from cold, copper sulfate (CuSO4), for malin and potassium permanganate (KMnO4). Exposure to permanganate was carried out in media containing serum, in serumdeficient media, and in saline solutions.

Cells were stressed for periods of 1-24 hours. During the final hour of incubation 35S-Methionine was added to the culture media. Cells were lysed by boiling in Laemmli sample buffer, and the lysate electrophoresed on 10-15% linear gradient polyacrylamide gels. After drying gels were placed in contact with radiographic film. The resultant autoradiograph depicted the pattern of proteins synthesized during the stress event. Lysates of heat-stressed cells were also used in two-dimensional electrophoresis and in monoclonal antibody studies designed to determine the degree of similarity between catfish hsps and those of other species.

Data collected demonstrated that catfish hepatocytes synthesize 17 different presumptive hsps. Different subsets of these proteins were induced by the various stressors. Furthermore, rates of synthesis of individual proteins (in response to a common stressor) varied during the 24-hour exposure period. Severe heat stress (temperature elevation from 28° to 40°C) resulted in induction of ten hsps, while less severe heat shock (elevation to 36°C) resulted in increased synthesis of two proteins. Recovery from cold shock (reduction from 28° to 12° or 18°C, followed by warming to original temperature) elicited increased synthesis of five proteins. Exposure to 0.2 mM CuSO4 and 20 ppm formalin also induced synthesis of nine and six proteins, respectively. Cells exposed to media not containing serum responded by increasing synthesis of four proteins.

Interestingly, permanganate (0.04 mM) appeared to suppress synthesis of two of these proteins when it was added to serum-deficient media. Two dimensional electrophoresis of heat-stressed cells demonstrated the presence of an hsp70 family of proteins, as well as a complex of five 40 kD proteins. Catfish hsp70 was also seen to be weakly and variably reactive with an anti-human hsp70 monoclonal antibody.

Data collected have helped to define the kinetics of the heat shock response mechanism under in vitro conditions. The next step is to determine if a correlation exists between these findings and studies involving intact fish. A possible experimental protocol would involve recovery of erythrocytes or leukocytes from stressed fish, immediate incubation with 35S-Methionine, and subsequent electrophoresis and autoradiography.

Cloning of fish hsp genes would also be of benefit because it would facilitate development of an ELISA or FA system of detection of these proteins.

Studies in humans have shown that hsp70 synthesis can be correlated with the severity of damage to selected tissues. Authors of these studies speculate that the degree of hsp production may also provide valuable prognostic information regarding the likelihood of recovery from a stress event. It is possible that hsp detection may likewise serve as an early, accurate and reliable means of detecting injurious (but unapparent) stressor-induced conditions in fish, and thus permit the institution of therapeutic modalities designed to ameliorate their harmful effects.

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Thomas G. Nemetz, BS, DVM, PhD

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