Induction of heat shock protein 72 by a nitric oxide donor in guinea-pig gastric mucosal cells
Introduction
Nitric oxide (NO) may be produced in small amounts within gastric mucous cells as a result of transient activation of a form of NO synthase similar to the neuronal enzyme (Price et al., 1996). Production of NO under such circumstances may increase mucus secretion by the cells (Brown et al., 1993). Mucous cells may also be continuously exposed to larger amounts of NO from exogenous sources. Thus nitrite in the gastric lumen may be converted to NO in the presence of acid (McKnight et al., 1997), and in gastritis a sustained release of NO by the inducible form of NO synthase present in neutrophils and macrophages (Mannick et al., 1996), and possibly in adjacent mucous cells (Brown et al., 1994) can be expected.
Short-term exposure to exogenous NO, derived from NO donors, damages freshly prepared suspensions of gastric mucosal cells (Tripp and Tepperman, 1996), but in hepatocytes longer exposure to exogenous NO induces expression of an heat shock protein which protects the cells from apoptosis induced by tumour necrosis factor α (Kim et al., 1997). Heat shock proteins play essential roles as molecular chaperons under stressed conditions by resolubilising protein aggregates and by facilitating refolding of denatured proteins (Welch, 1992). Thus the presence of heat shock proteins can protect guinea-pig gastric mucosal cells from damage by ethanol (Nakamura et al., 1991), and in macrophages heat shock proteins may promote resistance to damage by NO (Hirvonen et al., 1996).
Induction of the synthesis of heat shock proteins involves trimerisation of the transcription factor heat shock factor 1, and its transfer to the nucleus. This process may be induced by the depression of intracellular reduced thiol content (Huang et al., 1994), and NO has been found to reduce intracellular glutathione in suspensions of rat gastric mucosal cells (Wakulich and Tepperman, 1997). Consequently some measurements of reduced thiol content were made in this study.
Heat shock proteins in mammalian cells are separated into families according to their molecular mass. The most highly inducible protein following heat shock is heat shock protein 72 (HSP 72) (Welch, 1992), which was therefore selected for this investigation. The aims of the present work were to examine the long-term response of primary cultures of guinea-pig gastric mucous cells to exogenous NO and to determine whether there was induction of a protective response as exemplified by the presence of a heat shock protein.
Section snippets
Animals
Male Dunkin–Hartley guinea-pigs of 200–250 g body weight were obtained from Charles River, Margate, Kent, UK, and were fed on SDS Economy guinea-pig diet supplied by Lillico, Betchworth, Surrey, UK.
Materials
RPMI 1640 medium, foetal calf serum, antibiotics and amphotericin B were from Life Technologies, Paisley, UK. A monoclonal antibody, which recognises only HSP 72 from among the heat shock protein 70 family, was obtained from Amersham, Little Chalfont, UK. The bicinchoninic acid protein assay kit was
Effect of incubation with NO donors for 8 h on the presence of HSP 72, reduced thiol content and the viability of guinea-pig gastric cells
After 2 days of culture the cells preparations used for experiments contained >90% cells positive for periodic acid-Schiff reagent, and were therefore predominantly made up of mucous cells. Similar results have been obtained by others (Nakamura et al., 1991). Incubation with 1 mM S-nitroso-N-acetyl-penicillamine for 8 h produced an increase in the amount of HSP in guinea-pig gastric mucous cells (compare lanes 1 and 2 in Fig. 1, and also lanes 1 and 4 in Fig. 3, Fig. 4). The superoxide
Discussion
The central finding of this study is that an NO donor increased HSP 72 in guinea-pig gastric mucous cells. The inhibitory effect of the NO scavenger, carboxy-PTIO (Maeda et al., 1994), on the heat shock response is evidence that NO, or an NO-derived species, mediated the effect of S-nitroso-N-acetyl-penicillamine. The response was inhibited by actinomycin D and therefore probably involved increased transcription of the gene for HSP 72. Indeed in hepatocytes mRNA for HSP 72 was increased by
Acknowledgements
Clare Byrne was supported by a collaborative studentship involving Glaxo-Wellcome and the Medical Research Council, UK.
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