Porcine pulmonary arterial endothelial cells accumulated myo‐inositol and taurine, as well as betaine, during adaptation to hypertonic stress. The cells grew and maintained their normal morphology during culture in hypertonic (0.5 osmol (kg H2O)−1) medium that contained osmolytes such as betaine, myo‐inositol or taurine at concentrations close to reported physiological values. The cells did not grow well in hypertonic medium depleted of potential compatible osmolytes. After a few days, cell density decreased by about 50 % and many cells rounded up and detached from the plates, their nuclei showing clear apoptotic morphology. The caspase‐3 activity of the cells also increased dramatically under these conditions, but remained negligibly low when betaine and myo‐inositol were added to the medium. Addition of betaine and myo‐inositol to hypertonic medium depleted of compatible osmolytes increased the number of colonies remaining after 12 days of culture; with each solute at 30–100 μmol l−1 the number increased about sixfold. In the absence of compatible osmolytes, increased mRNA levels and corresponding activities of betaine/γ‐aminobutyric acid transporter (BGT1) and sodium/myo‐inositol transporter (SMIT) induced by hypertonicity remained high after 72 h incubation, whereas they were down regulated in the presence of betaine and myo‐inositol. Similarly, the down regulation of the amino acid System A transporter (ATA2) was markedly slowed in the absence of compatible osmolytes. We conclude that these compatible osmolytes at concentrations close to physiological values enable the endothelial cells to adapt to hypertonic stress, protecting them from apoptosis, and also modulate the adaptation process.
Various solutes were tested to see if they could modify the responses of SV-3T3 cells to hyperosmotic (0.5 osM) conditions, which cause an inhibition of general cell protein synthesis and of the rate of cell proliferation, coupled with an induction of amino acid transport activity. The added solutes were glycerol, proline, taurine, betaine, dimethylglycine and sarcosine. Of these, betaine produced the most dramatic and consistent effects. Addition of 10-25 mM-betaine to the hyperosmotic medium largely prevented the 90% inhibition of cell proliferation that occurred in its absence. Whether it was added initially or after the cells were exposed to hyperosmotic medium, 25 mM-betaine also converted a 50% recovery of the rate of protein synthesis into 100%. Similarly, the same concentrations of betaine prevented a 30% decrease in cell volume and decreased the induction of amino acid transport via system A by 73%. Lower concentrations of betaine produced smaller but still significant changes in these functional responses. With chick-embryo fibroblasts, under identical hyperosmotic conditions, 25 mM-betaine completely counteracted a 75% inhibition of the rate of protein synthesis. At present it is not clear how betaine modulates these effects of hyperosmolarity on cell functions.
We studied the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. In 0.5 osmol/kgH(2)O medium, initial cell shrinkage was followed by a regulatory volume increase (RVI), complete after 1 h, concomitant with an increase in cellular K(+) content. Then the activity of amino acid transport System A increased, accompanied by an accumulation of ninhydrin-positive solutes (NPS), reaching a peak at approximately 6 h. The subsequent decline in System A activity was paralleled by an induction of the betaine-GABA transporter (BGT-1), detected as increases of BGT-1 mRNA and of transport activity, which peaked at approximately 24 h. Inhibitors of transcription or translation prevented induction of both transport activities. The increased expression of BGT-1, which involved activation of "tonicity-responsive enhancer," was inhibited by 5 mM extracellular betaine. Cellular K(+) concentration gradually declined after the accumulation of NPS and during the induction of BGT-1. This very effective adaptation to hypertonicity suggests it has a physiological role.
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