Response of human fibroblasts to hypertonic stress. Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids

Abstract

Regulatory volume increase (RVI) has been studied in cultured human fibroblasts (CHF) incubated in a complete hypertonic growth medium (400 mosmol/kg). After the initial cell shrinkage induced by hypertonic treatment, cells recover their volume almost completely within 3 h. This RVI response is associated with a marked increase of the cell content of free amino acids. The cell content of potassium increases only slightly. Chromatographic analysis of the intracellular amino acid pool shows that the RVI-associated increase in cell amino acids is mainly a result of changes in the L-glutamine content. The intracellular accumulation of the analog 2-methylaminoisobutyric acid, a specific substrate of transport system A, is increased in CHF undergoing RVI. Hypertonic treatment causes an immediate and sustained cell hyperpolarization, as demonstrated by changes in the trans-membrane distribution ratio of L-arginine and in the fluorescence of the potential-sensitive dye bis-1,3-diethylthiobarbiturate-trimethineoxonol. Because of cell hyperpolarization, at the end of RVI the trans-membrane gradient of the sodium electrochemical potential is higher than that of the control. The increase in the extracellular potassium concentration ([K+]out = 40 mM) abolishes the hyperpolarization induced by hypertonic treatment and delays volume recovery. Cycloheximide suppresses RVI at a high but not at physiologic [K+]out. It is proposed that CHF counteract hypertonic shrinkage through an enhanced accumulation of substrates of transport system A sustained, initially, by an increase in the energy available for transport and, subsequently, also by the synthesis of new site A carriers.