A simple procedure has been established for controlling and measuring the pH of media in which the bicarbonate-carbonic acid system is the predominant buffer. The HCO-3 concentration was maintained at 22.5 mM and the H2CO3 concentration was varied by equilibrating the media with 0.5 to 40 per cent CO2 in air. The curve relating extracellular pH to 3 day cell growth was similar for glass-attached HeLa and Chang liver cells. Maximum growth occurred over a pH range of 7.38 to 7.87. Cell growth declined precipitously on the alkaline side and more gradually on the acid side of the optimal pH range. Comparable pH growth curves were also obtained with newly isolated cells from rat liver and skeletal muscle. It was shown that the effect of pH on growth was independent of the CO2 concentration and that the essential nutrients in the medium were stable over the pH range studied. Although alkalosis depressed the 3 day cell population, cells exposed to a pH of 8.0 to 8.2 grew at the maximal rate for the first 12 to 24 hours. Growth then ceased abruptly and the cells entered a steady state with respect to net protein synthesis. This was followed by cytoplasmic retraction and cell death. Increasing the concentrations of calcium or magnesium in the medium failed to prevent the effects of alkalosis. Moreover, the increase in CO-3 concentration of the media and the concomitant decrease in Ca++ ion concentration that occur at high pH were eliminated as determining factors in the growth failure and death. While acidosis had a less pronounced effect on the 3 day cell population, its effect on the growth rate was immediate. The increase in cell generation time was proportional to the H+ ion concentration. In each of the cell lines studied, acidosis was accompanied by a striking increase in the number of cytoplasmic perinuclear granules. These granules which stain supravitally with Janus green are extracted from fixed cells with lipid solvents. They maintain their identity in cell homogenates and may be isolated from the other subcellular structures by differential centrifugation; at 100,000 g they form a distinct layer at the top of the supernatant fraction. On the basis of their physical and chemical properties, these granules have been called lipid-rich particles. The accumulation of lipid-rich particles in acidosis was independent of the growth rate and the CO2 concentration.
THE EFFECT OF pH ON GROWTH, PROTEIN SYNTHESIS, AND LIPID-RICH PARTICLES OF CULTURED MAMMALIAN CELLS
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Cosmo G. Mackenzie, Julia B. Mackenzie, Paul Beck; THE EFFECT OF pH ON GROWTH, PROTEIN SYNTHESIS, AND LIPID-RICH PARTICLES OF CULTURED MAMMALIAN CELLS . J Biophys and Biochem Cytol 1 January 1961; 9 (1): 141–156. doi: https://doi.org/10.1083/jcb.9.1.141
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