A mechanism of glycine and alanine cytoprotective action: Stimulation of stress-induced HSP70 mRNA

A mechanism of glycine and alanine cytoprotective action: Stimulation of stress-induced HSP70mRNA. Studies done both in vitro and in vivo have shown that glycine and alanine protect kidney cells from stress injury. However, the mechanism(s) of this cytoprotection is unknown. Our aim was to test the hypothesis that the cytoprotective action is in part due to stimulation of gene(s) expression encoding stress protein synthesis. Experiments were carried out using heat shock as a model for stress in the opossum kidney cell line (OK cells). The induction of HSP70 mRNA was evaluated in cell monolayers exposed to 45°C for 15 minutes followed by a recovery period at 37°C for either 0.5, 1, 2, 3, 4, 6 or 24 hours. The results demonstrate that the maximum level of HSP70mRNA occurred at ≈ three hours after heat treatment. Although the mRNA levels declined thereafter, appreciable amounts were still seen even 24 hours after heat-shock. To examine the effect of glycine or alanine on HSP70mRNA levels and on the synthesis of stress protein, cultures were preincubated for 30 minutes with Krebs-Henseleit buffer, pH 7.4, supplemented with either 1, 2, 5 or 10mM glycine or alanine, or with no added amino acids. Comparative studies were performed with 10mM glutamate, aspartate, arginine or leucine. Following preincubation, cultures were heat-shocked (45°C for 15min) and then reincubated at 37°C for three hours. Both glycine and alanine enhanced the level of HSP70mRNA and the synthesis of 72,73kDa stress proteins, but neither amino acid induced HSP70mRNA without concomitant heat treatment. Glutamate, aspartate, leucine and arginine had no enhancing effect, however, their inclusion in the incubation medium induced heat-shock-like response without heat treatment. The increased level of HSP70 mRNA and the synthesis of stress protein in the presence of glycine or alanine were associated with decreased cellular LDH release, suggesting greater thermotolerance of the cultured cells. Intracellular ATP levels declined following heat shock in all experiments. Supplementation of the medium with glycine or alanine did not alter this stress-induced reduction of intracellular ATP, supporting a previous suggestion that the cytoprotective action of glycine and alanine is independent of cellular ATP levels. The current data suggest a functional role for glycine and alanine in the stimulation of gene(s) expression encoding for stress protein(s) synthesis, and in protecting cells against stress da